Chimeric antigen receptor comprising interleukin-15 intracellular domain and uses thereof

ABSTRACT

Provided herein are chimeric antigen receptors (CARs) comprising an antigen binding domain (e.g., CD19, CD30, GD2, etc.), transmembrane domain (e.g., CD28), and a cytoplasmic domain (e.g., CD27, 4-1BB, etc.). In some aspects, the disclosure relates to use of the CARs in T cells, compositions, kits and methods.

RELATED APPLICATIONS

This application is a Divisional of U.S. Ser. No. 15/552,071, filed Aug.18, 2017, entitled “CHIMERIC ANTIGEN RECEPTORS AND USES THEREOF”, whichis a National Stage Application of PCT/US16/18716, filed Feb. 19, 2016,entitled “CHIMERIC ANTIGEN RECEPTORS AND USES THEREOF”, which claims thebenefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Ser.No. 62/118,080, filed on Feb. 19, 2015, entitled “CHIMERIC ANTIGENRECEPTOR COMPRISING INTERLEUKIN-15 RECEPTOR INTRACELLULAR DOMAIN ANDUSES THEREOF”, 62/134,444, filed on Mar. 17, 2015, entitled “CHIMERICANTIGEN RECEPTORS THAT TARGET GD2”, 62/152,792, filed on Apr. 24, 2015,entitled “CHIMERIC ANTIGEN RECEPTORS THAT TARGET CD30 AND USES THEREOF”,and 62/165,836, filed on May 22, 2015, entitled “CHIMERIC ANTIGENRECEPTORS THAT TARGET GD2”, the entire contents of each applicationwhich are incorporated herein by reference.

BACKGROUND

Chimeric Antigen Receptors (CARs) are engineered T cell receptorsdisplaying specificity against target antigens based on a single chainFV (scFv) antibody moiety. Although CARs show promise as therapeuticagents, several challenges (e.g., the induction of antigen-specifictoxicities targeting normal tissues expressing the target-antigen, andthe extreme potency of CAR-T cell treatments resulting inlife-threatening cytokine-release syndromes) limit their use in aclinical context. Therefore, there is a need to develop safer and moreclinically effective CARs.

SUMMARY

In some aspects, the disclosure relates to chimeric antigen receptors(CARs) comprising an antigen binding domain (e.g., anti-CD19, anti-CD30,anti-GD2), and/or a cytoplasmic domain of a interleukin(IL)-15-receptorα, and/or a chimeric cytoplasmic domain including a CD27 cytoplasmicdomain fused to a 4-1BB cytoplasmic domain. Use of the CARs in T cells,compositions, kits and methods is also contemplated by the disclosure.

Aspects of the disclosure relate to CARs comprising a cytoplasmic domainof a interleukin(IL)-15-receptor α, and uses of such CARs to produce CART-cells (CARTs) and/or CAR-modified immune cells such as NK (naturalkiller) cells, which can be used in various methods, such as treatmentmethods, or compositions.

As described herein, it was found that including the cytoplasmic domainof IL-15-receptor α (IL-15Rα) in a CAR construct resulted in CARTs withgreater expansion potential upon antigen stimulation and higher killingefficacy. Further, the CARTs maintained killing efficacy even onrepetitive addition of an excess of target cells. The CARTs alsoproduced increased amounts of effector cytokines, as determined byintracellular staining and flow cytometry based bead assays.

Accordingly, aspects of the disclosure relate to a chimeric antigenreceptor (CAR) comprising an antigen binding domain; a transmembranedomain; and a cytoplasmic domain containing an interleukin 15-receptor α(IL-15Rα) cytoplasmic domain. In some embodiments, the transmembranedomain is a CD28 or CD8 transmembrane domain. In some embodiments, thecytoplasmic domain further comprises a CD3zeta signal transductiondomain. In some embodiments, the cytoplasmic domain further comprises aCD27 signaling domain. In some embodiments, the antigen binding domainis a single-chain variable fragment (scFv). In some embodiments, theantigen binding domain is specific for CD19.

Aspects of the disclosure relate to CARs comprising a chimericcytoplasmic domain including a CD27 cytoplasmic domain fused to a 4-1BBcytoplasmic domain, and uses of such CARs to produce CARTs and/orCAR-modified immune cells such as NK (natural killer) cells, which canbe used in various methods, such as treatment methods, or compositions.

In some aspects, the disclosure relates to a chimeric antigen receptor(CAR) comprising: an antigen binding domain; a transmembrane domain; anda cytoplasmic domain containing a CD27 intracellular domain.

In some embodiments, the transmembrane domain is a CD28 or CD8transmembrane domain. In some embodiments, the cytoplasmic domainfurther comprises a 4-1BB intracellular domain. In some embodiments, thecytoplasmic domain further comprises a CD3zeta signal transductiondomain. In some embodiments, the cytoplasmic domain further comprises aniCasp9 domain and/or a FKBP domain. In some embodiments, the antigenbinding domain is a single-chain variable fragment (scFv).

In some embodiments, a CAR comprises a CD28 transmembrane domain,cytoplasmic domain comprising a CD27 intracellular domain and a 4-1BBintracellular domain, and a CD3zeta signal transduction domain. In someembodiments, a CAR further comprises an iCasp9 domain and/or a FKBPdomain.

In some embodiments, a CAR further comprises a first spacer between theCD28 transmembrane domain and the cytoplasmic domain comprising a CD27intracellular domain and a 4-1BB intracellular domain, and a secondspacer between the cytoplasmic domain comprising a CD27 intracellulardomain and a 4-1BB intracellular domain and the CD3zeta signaltransduction domain. In some embodiments, the CAR further comprises athird spacer between the CD3zeta signal transduction domain and theiCasp9 domain and/or a FKBP domain. In some embodiments, the antigenbinding domain of a CAR is specific for CD30.

Aspects of the disclosure relate to CARs comprising an antigen-bindingdomain specific for Disialoganglioside 2 (GD2), and uses of such CARs toproduce CAR-modified immune cells such as T cells (also referred toherein as CARTs) or NK (natural killer) cells, which can be used invarious methods, such as treatment methods, or compositions. In someaspects of the disclosure, such CARs are used to treat retinoblastoma orosteosarcoma, such as a juvenile subject having retinoblastoma orosteosarcoma.

As described herein, it was found that GD2-specific CAR-expressing Tcells could kill both osteosarcoma and retinoblastoma cells, both ofwhich express GD2. As a result, it is expected that GD2-specific CARswill be useful for treatment of both osteosarcoma and retinoblastoma.

Aspects of the disclosure relate to a chimeric antigen receptor (CAR)comprising an antigen binding domain specific for GD2; a transmembranedomain; and a cytoplasmic domain containing one or more (e.g., one, two,or three) of a CD27 signaling domain, a 4-1BB intracellular domain, anda CD3zeta signal transduction domain. In some embodiments, thetransmembrane domain is a CD28 or CD8 transmembrane domain. In someembodiments, the antigen binding domain is a single-chain variablefragment (scFv).

Other aspects of the disclosure relate to a nucleic acid comprising asequence that encodes the CAR of any one of the above embodiments or asotherwise described herein.

Yet other aspects relate to an immune cell comprising a CAR of any oneof the above embodiments or as otherwise described herein and/or anucleic acid of any one of the above embodiments or as otherwisedescribed herein. In some embodiments, the immune cell is a T cell or NKcell. In some embodiments, the immune cell is a T cell.

Other aspects of the disclosure relate to a composition comprising aplurality of the immune cell of any one of the above embodiments or asotherwise described herein. In some embodiments, the compositioncomprises a pharmaceutically acceptable carrier.

Another aspect of the disclosure relates to a method of generating aplurality of CAR-modified immune cells, the method comprisingintroducing a lentiviral vector comprising a nucleic acid of any one ofthe above embodiments or as otherwise described herein into a pluralityof immune cells. In some embodiments, the immune cells are T cells.

Other aspects of the disclosure relate to a method of treating a subjecthaving a disease, the method comprising administering an immune cell ofany one of the above embodiments or as otherwise described herein, thecomposition of any one of the above embodiments or as otherwisedescribed herein, or the plurality of immune cells produced by a methodof any one of the above embodiments or as otherwise described hereininto a subject having a disease or at risk of having a disease.

In some embodiments, the disease is cancer, an autoimmune disease or aninfection. In some embodiments, the disease is cancer. In someembodiments, the disease is a CD30+ cancer. In some embodiments, thecancer is osteosarcoma or retinoblastoma.

In some embodiments, the method further comprises administering a PD-L1or PD1 inhibitor to the subject.

Other aspects of the disclosure relate to a method of treating a subjecthaving cancer, the method comprising administering (a) an immune cellexpressing a CAR that targets GD2 and (b) a PD-L1 or PD1 inhibitor to asubject having cancer. In some embodiments, the cancer is osteosarcomaor retinoblastoma. In some embodiments, the immune cell is a T cell. Insome embodiments, the immune cell is a plurality of immune cells in acomposition.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure, which can be better understood by reference to one or moreof these drawings in combination with the detailed description ofspecific embodiments presented herein. It should be appreciated that ingreyscale versions of the drawings, GFP fluorescence appears as areas oflighter shading (e.g., white shading)

FIG. 1 is a diagram showing an exemplary Chimeric Antigen Receptor (CAR)basic structure including the antigen binding scFv domain and thevarious co-stimulatory domains.

FIG. 2 is a diagram showing an exemplary simple killing assay—Co-cultureof green fluorescent protein wasabi (GFP)-labeled target with the CARTeffector cells and flow cytometry after annexinV/PI staining.

FIG. 3 is a series of plots and two photographs showing that exemplarylentiviral gene transfer into human T cells is very high. The X-axis ineach plot shows CD3 staining and the Y-axis in each plot shows GFPstaining.

FIG. 4 is a series of photographs showing exemplary primary T cellkilling efficiency demonstrated by disappearance of the green(fluorescent) target cells as seen under the fluorescent microscope.Both bright-field and fluorescent images are shown for each sample.

FIG. 5A is a series of plots and a graph showing exemplary healthy donorderived CART cells—Flow data of Killing in terms of % early apoptoticcells with annexinV positive and PI negative staining seen after 1-2hours of co-culture.

FIG. 5B is two graphs showing exemplary % total cell death (top graph)for each CAR and % specific lysis (bottom graph) for each CAR. This wasdone in triplicate wells to acquire mean values and statisticalsignificance obtained using student t test (p<0.05 consideredsignificant). *P<0.01 (comparing with No CAR), #P<0.01 (comparing with acontrol CART), {circumflex over ( )}P<0.05 (comparing 153z with 19z and273z).

FIG. 6A is a series of graphs showing exemplary flow data of killingafter overnight incubation in terms of % early and late apoptotic cells(both annexinV+PI− and double positive for AnnexinV and PI)

FIG. 6B is a series of plots and graphs showing exemplary long termkilling after 3 days of co-coculture of patient derived CART cells withgreen (fluorescent) target cells. Killing was indicated by disappearanceof the green target cells.

FIG. 6C is a series of photographs and a graph showing exemplary longterm killing after 3 days of co-coculture of patient derived CART cellswith green (fluorescent) target cells. Killing was indicated bydisappearance of the green (fluorescent) target cells.

FIGS. 7A and 7B show a series of plots and graphs of exemplary patientALL cells labeled with Calcein AM and patient derived CART cellsincubated in 5:1 ratio. Flow data shows killing at 1 hour (FIG. 7A) and1 day (FIG. 7B) in terms of early apoptosis with % annexinV positive andPI positive cells.

FIG. 8 is a series of plots showing exemplary retargeting of CARTeffector cells with green (fluorescent) target. Killing seen asindicated by disappearance of target in flow cytometry.

FIG. 9 is a series of plots and a graph showing exemplary CART cellproliferation by a CFSE method. Proliferation peak and % seen after CARTcells stimulation with target cells.

FIGS. 10A and 10B are a series of graphs showing exemplary intracellulareffector function analysis flow data of IL-2, IFN-g and CD107a producedafter the CART cells are co-cultured with the target cells—RS4-11.Positive control (PC) is PMA/ionomycin stimulation.

FIG. 10C is a graph showing the exemplary data summarized from FIGS. 10Aand 10B in bar graph form. Maximum IL-2, IFN-g and CD107a release wasseen with 153z CART cells.

FIG. 11 shows flow cytometry analysis of CD30 surface expression of aprimary B cell tumor line.

FIG. 12 shows an illustration of the lentiviral vector system.

FIG. 13 shows diagrams illustrating embodiments of CAR molecules,including one embodiment of a 4th generation CD30-CAR structure andcomponents.

FIG. 14 shows an illustration of a rapid CAR T target killing assay usedfor the evaluation of CAR functions.

FIG. 15 shows data related to a target killing assay of two differentCD30-CAR T cells based on 5F11 and AC10 scFv CD30-CARs.

FIG. 16 shows flow cytometry target killing assay of two differentCD30-CAR T cells based on 5F11 and AC10 scFv CD30-CARs.

FIG. 17 shows the clinical course of a Hodgkin's lymphoma patienttreated with CD30-CAR T cells. The infusion time point was depicted byarrows. Three batches of CAR T cells were prepared and infused asindicated. CAR-positive T cells were detected by qPCR detection of CARDNA in the genome of blood cells. Clinical evidence of tumor regressionwas evident at day 40 after the first infusion.

FIG. 18 shows data related to detection of IL-6 cytokine response afterCD30 CAR T infusion.

FIG. 19 shows data related to detection of IFN-gamma cytokine responseafter CD30 CAR T infusion.

FIGS. 20A-20C shows graphs of exemplary GD2 surface expression inosteosarcoma (OS) cell lines. Flow cytometry histogram overlays showsurface expression of GD2 in (FIG. 20A) HOS (FIG. 20B) U2OS and (FIG.20C) OS156 cells. The dark line represents GD2 positive and the shadowedgrey line represents isotype control. Inner number indicates percentpositive population and mean fluorescence intensity (MFI) of thepositive population

FIG. 21A is a schematic showing an exemplary CAR that targets GD2binding to a cell expressing GD2.

FIG. 21B is a schematic that shows embodiments of CAR molecules,including one embodiment of GD2 CAR construction and 4th generation CARconfigurations.

FIG. 22 is a diagram that shows an exemplary CAR-JK/T cell killingassay.

FIGS. 23A-23D are a series of plots and graphs that show short term GD2JK killing. JK cells were used for CAR construction as a preliminarydetermination of GD2 CAR killing ability. FIG. 23A) Representative dotplots from short term killing of U2OS cells by GD2 CAR-JK cells (E:Tratio=2:1). Effecter and target cells are separated by FITC signal, FITCpositive target cells (upper panels) were gated and determinedpercentage of death cell by Annexin V versus PI plots (lower panels).FIG. 23B) Percent specific lysis of U2OS by GD2 CAR-JK cell waspresented in comparison with control CAR-JK cell, * indicates asignificant difference (p=0.0049). FIG. 23C) Representative dot plotsfrom short term killing of HOS cells by GD2 CAR-JK cells (E:T ratio=2:1)showing percentage of FITC positive target cells (upper panels) andAnnexin V and/or PI positive target cells (lower panels). FIG. 23D)Percent specific lysis of HOS by GD2 CAR-JK cell shows higher but notsignificant different compare to control CAR-JK cells (p=0.078; NS=notsignificant different).

FIGS. 24A-24G show exemplary CAR-Primary T cell killing of OS target.The killing ability of GD2 CAR in primary T cells were determined. FIG.24A) Flow cytometry analysis of U2OSw cells by GD2 CAR-T cells afterco-culture at E:T ratio of 4:1, 2:1, 1:1, 1;2, * indicates significantdifferent from controls (p<0.05). FIG. 24B) Percent specific lyses ofU2OSw cells after 3 day co-culture, * indicates significant differentfrom control T cells (p<0.05). FIG. 24C) Flow cytometry analysis of HOSwcells by GD2 CAR-T cells after co-culture at E:T ratio of 4:1, 2:1, 1:1,1;2, * indicates significant different from controls (p<0.05). FIG. 24D)Percent specific lyses of HOSw cells after 3 day co-culture, * indicatessignificant different from control T cells (p<0.05). FIG. 24E) Percentspecific lyses of OS156 cells after 3 day co-culture, * indicatessignificant different from control T cells (p<0.05). FIG. 24F) Targetcells (green, fluorescent) and effecter cells (no color) underfluorescence microscope after 4 days co-culture. FIG. 24G) Target cells(green, fluorescent) and effecter cells (no color) under fluorescencemicroscope after 3, 7 or 12 days co-culture.

FIGS. 25A-25D show exemplary PDL-1 expression in OS cells afterincubation with CAR T cells. FIG. 25A) and FIG. 25B) Flow cytometryhistogram overlays show surface expression of PD-L1 in U2OS and HOScell. The shadowed area represents isotype control. Inner numberindicates percent positive population and MFI of the positivepopulation. FIG. 25C) and FIG. 25D) MFI analysis of PD-L1 expression inOS cells after CAR T co-culture.

FIGS. 26A-26F show exemplary PD-1 expression in CAR T cells afterincubation with OS cells. PD1 expression in primary CAR T cells after 1day co-culture with target cells (E:T ratio=2:1), U2OSw (FIGS. 26A-26B)and HOSw (FIGS. 26D-26E). A bar graph displays fold increase of PD1expression from background expression level. The number above the barsshow fold increase of PD1 expression. FIG. 26C) and FIG. 26F) IncreasedCAR T cell death after PD-1 up-regulation. Percent CAR T cell deathafter 1 day co-culture with U2OSw (C) and HOSw (D) cells, # indicatessignificant different at p<0.05.

FIG. 27A is a series of photographs that shows immunohistochemistryanalyses of GD2 expression in tumor samples from RB patients.Paraffin-embedded retinal tumor samples were stained for GD2 expressionby using antibody against human GD2 protein.

FIG. 27B is a series of graphs that shows surface staining of GD2expression on Y79 retinoblastoma (RB) cell line. Flow cytometricstaining for GD2 and CD19 proteins (black line) or an IgG control(filled histogram) on the surface of Y79 RB cells. Upper right numberindicates cell count and the normalized mean fluorescence intensities(nMFI) are shown in parenthesis.

FIG. 28A is a diagram of exemplary GD2 CAR constructs.

FIG. 28B is a diagram showing an exemplary cell killing assay for RBcells. Y79 RB cells were engineered to express wasort, a greenfluorescent protein (GFP), using lentiviral transduction. Y79 RB wasortcells were co-cultured with control T cells or CD19 T cells, or GD2 CART cells. After 24 hr of co-cultivation, apoptosis of tumor cells wereexamined by using AnnexinV/PI staining and analyzed by flow cytometry.

FIGS. 29A-29D show an exemplary short-term killing assay on Y79 RB usingCAR T-cells. FIG. 29A shows analysis of cell death by using AnnexinV/PIstaining. After 24 hr of co-culture experiment, the suspension cellswere harvested and stained with AnnexinV-PE/PI dye followed by flowcytometric analysis. FIG. 29B shows the percent cell death at 1 dayafter co-culture. FIG. 29C shows the % of specific lysis. FIG. 29D is aseries of photographs showing fluorescence microscopic monitoring oftumor cell lysis. Y79 RB wasort cells (1×105) were cultured with controlT cells or CD19 CAR T cells, or GD2 CAR T cells at the effector/targetratio (1:1) for 24 h, and the lysis of tumor cells were monitored underfluorescence microscope.

FIG. 30 is a series of photographs that shows exemplary 2^(nd) roundkilling of target RB cells by GD2-CAR T cells at day 6 or day 14 ofco-culture.

FIGS. 31A and 31B are a series of graphs that show down-regulation ofGD2 in RB tumor upon GD2-CAR T targeting.

FIG. 31C is a series of graphs that shows upregulation of PD-L1expression upon GD2-CAR T cell targeting.

FIG. 31D is a series of graphs that shows down-regulation of GD2expression upon GD2-CAR T cell targeting.

DETAILED DESCRIPTION

In some aspects, the disclosure relates to chimeric antigen receptors(CARs) comprising an antigen binding domain (e.g., anti-CD19, anti-CD30,anti-GD2), and/or a cytoplasmic domain of a interleukin(IL)-15-receptorα, and/or a chimeric cytoplasmic domain including a CD27 cytoplasmicdomain fused to a 4-1BB cytoplasmic domain. Use of the CARs in T cells,compositions, kits and methods is also contemplated by the disclosure.

The invention relates, in some embodiments, to chimeric antigenreceptors (CARs) and uses thereof in T cells (e.g., to make CAR Tcells), methods, nucleic acids, compositions, kits and the like. CARsare molecules that combine antibody-based specificity for a desiredantigen (e.g., tumor antigen) with a T cell receptor-activatingintracellular domain to generate a chimeric protein that exhibits aspecific anti-tumor cellular immune activity. It should be appreciatedthat, in some embodiments, exemplary, non-limiting arrangements of CARsare described from left to right, N-terminus to C-terminus of the CAR.

In some aspects, the disclosure relates to CARs having an interleukin15-receptor α (IL-15Rα) cytoplasmic domain. CARs containing this domainhad several advantageous and surprising features including greaterexpansion potential upon antigen stimulation, higher killing efficacy,maintained killing efficacy upon repetitive addition of excess targetcells, and an increased amount of effector cytokines compared to CARsnot containing this domain.

In some embodiments, the present invention relates, in part, to the useof T cells genetically modified to stably express a desired CAR, e.g.,containing a IL-15Rα cytoplasmic domain, containing a CD30 antigenbinding domain, containing a GD2 antigen binding domain, etc. T cellsexpressing a CAR are referred to herein as CAR T cells, CARTs, or CARmodified T cells. Preferably, the cell can be genetically modified tostably express an antibody binding domain on its surface, conferringnovel antigen specificity that is MHC independent. In some instances,the T cell is genetically modified to stably express a CAR that combinesan antigen recognition domain of a specific antibody with antransmembrane domain and a cytoplasmic domain into a single chimericprotein. In some embodiments, two CAR proteins dimerize (e.g., formhomo- or heterodimers) in vivo.

In some embodiments, a CAR comprises an antigen binding domain, atransmembrane domain, a cytoplasmic domain comprising an IL-15Rαcytoplasmic domain, optionally further comprising a CD3 zeta (CD3z)signaling domain and/or a CD27 signaling domain. In some embodiments,the arrangement of the elements of the CAR is selected from one of thefollowing exemplary, non-limiting arrangements (from N-terminus toC-terminus):

scFv-CD28-IL-15Rα-CD3zscFv-CD28-CD27-IL-15Rα-CD3zscFv-CD28-(4-1BB)-CD27-IL-15Rα-CD3zscFv-CD8-CD27-IL-15Rα-CD3zscFv-CD8-(4-1BB)-CD27-IL-15Rα-CD3z

In some aspects, the disclosure relates to CARs having a CD30 antigenbinding domain and an intracellular domain comprising a 4-1BBintracellular domain and a CD27 intracellular domain. In someembodiments, the CARs further comprise a self-destructive domain (e.g.,an iCasp9-FKBP domain) that further improves safety. CARs containingthese domains had several surprising features including an acceptablesafety profile, and mediate effective killing of CD30⁺ cancer cells bothin vitro and in vivo.

In some embodiments, a CAR (e.g., CAR having a CD30 antigen bindingdomain) comprises an antigen binding domain, a transmembrane domain, acytoplasmic domain comprising a 4-1BB intracellular domain and a CD27intracellular domain, optionally further comprising a CD3 zeta (CD3z)signaling domain, and/or an apoptosis-inducing iCasp9-FKBP domain. Insome embodiments, the arrangement of the elements of the CAR is selectedfrom one of the following exemplary, non-limiting arrangements (fromN-terminus to C-terminus):

scFv-CD28-(4-1BB)-CD27-CD3z-iCasp9-FKBPscFv-CD28-CD27-(4-1BB)-CD3z-iCasp9-FKBPscFv-CD8-(4-1BB)-CD27-CD3z-iCasp9-FKBPscFv-CD8-CD27-(4-1BB)-CD3z-iCasp9-FKBP

In some aspects, the disclosure relates to the discovery that GD2 wasexpressed on both osteosarcoma and retinoblastoma cells and thatGD2-specific CAR-expressing T cells could kill both osteosarcoma andretinoblastoma cells. As a result, it is expected that such CARs (e.g.,GD2-specific CARs) will be useful for treatment of both osteosarcoma andretinoblastoma. In some embodiments, GD2-specific CARs contain anantigen binding domain (such as an scFV) specific for GD2. In someembodiments, the arrangement of the elements of the CAR (e.g.,GD2-specific CAR) is selected from one of the following exemplary,non-limiting arrangements (from N-terminus to C-terminus):

GD2scFv-CD28-(4-1BB)-CD27-CD3z GD2scFv-CD8-CD28-CD3zGD2scFv-CD28-(4-1BB)-CD27-CD3z-Casp9-FKBPGD2scFv-CD8-CD28-CD3z-Casp9-FKBP

A CAR molecule may also include several hinge elements and/or spacersequences (such as between individual domain elements). In someembodiments, the spacer and/or hinge sequences of the CAR are selectedfrom one or more of the following exemplary sequences:

(SEQ ID NO: 1) GGGGS, (SEQ ID NO: 2) GGGGSGGGGS, (SEQ ID NO: 3)(GGGGS) × 3, (SEQ ID NO: 4) GSTSGGGSGGGSGGGGSS, (SEQ ID NO: 5)GSTSGSGKPGSSEGSTKG, (SEQ ID NO: 6) GGGGSGGG, (SEQ ID NO: 7)VEPKSCDKTHTCPPCP, (SEQ ID NO: 8) LDPKSSDKTHTCPPCP, (SEQ ID NO: 9)VEPKSPDKTHTCPPCP, or (SEQ ID NO: 10) LDKTHTCPPCPP.

In some embodiments, the present invention relates, in part, to a CARthat incorporates a series of domains that provide different functionalaspects that may synergistically work together to improve efficacy. Forexample, the CAR may include one or more of: an antigen binding domain,a hinge domain, an antigen co-signaling domain that stimulates activity(e.g., an IL-15Rα cytoplasmic domain, a 4-1BB cytoplasmic domain, oranother cytoplasmic domain), a survival domain that increases T-cell orimmune effector cell survival, a T-cell or immune effector cell memorydomain, and an effector activating domain. Alternatively, the CAR mayfurther include a domain that induces safety (e.g., anapoptosis-inducing iCasp9-FKBP domain).

In some embodiments, a CAR of the invention comprises an extracellulardomain having an antigen binding domain, a transmembrane domain, and amulti-functional cytoplasmic domain. In some embodiments, the CARcomprises a fully human antibody or antibody fragment. In someembodiments, the transmembrane domain that naturally is associated withone of the domains in the CAR is used. In another embodiment, thetransmembrane domain can be selected or modified by amino acidsubstitution to avoid binding of such domains to the transmembranedomains of the same or different surface membrane proteins to minimizeinteractions with other members of the receptor complex.

In some embodiments, the CAR T cells or other CAR-modified immune cells(e.g., CAR-modified NK cells) of the invention are generated byintroducing a lentiviral vector comprising a nucleic acid that encodes adesired CAR into T cells or other immune cells (e.g., into NK cells). Insome embodiments, the lentiviral vector comprises a nucleic acid thatencodes a CAR comprising an antigen binding domain (e.g., that targetsCD19), a transmembrane domain, and a cytoplasmic domain. In someembodiments, the CAR T cells or other CAR-modified immune cells (e.g.,NK cells) of the invention are able to replicate in vivo resulting inlong-term persistence that can lead to sustained tumor control. In someembodiments, the CAR T cell or other CAR-modified immune cells of theinvention can be generated by transfecting a transposon or RNA encodingthe desired CAR, into the T cells or other immune cells. In someembodiments, the CAR is transiently expressed in the geneticallymodified CAR T cell or other CAR-modified immune cells.

In some embodiments, the invention relates to administering agenetically modified immune cell (e.g., a genetically modified T cell orNK cell) expressing a CAR for the treatment of a patient having canceror at risk of having cancer, or having an autoimmune disease or at riskof having an autoimmune disease, e.g., using lymphocyte infusion. Insome embodiments, autologous lymphocyte infusion is used in thetreatment. In some embodiments, autologous PBMCs are collected from apatient in need of treatment and immune cells (e.g., T cells or NKcells) are activated and expanded using the methods described herein andknown in the art and then infused back into the patient.

In some embodiments, the cancer is a CD30+ cancer (e.g., a cancerexpressing CD30). Examples of cancers expressing CD30 include Hodgkin'slymphoma (HL), anaplastic large cell lymphoma (ALCL), diffuse large Bcell lymphoma, primary effusion lymphoma, adult T-cellleukemia/lymphoma, mycosis fungoides, extranodal natural killer/T-celllymphoma and peripheral T/NK cell lymphoma. In some embodiments thecancer is a GD2-expressing cancer, such as retinoblastoma orosteosarcoma.

Exemplary Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used.

“Activation”, as used herein, refers to the state of a T cell that hasbeen sufficiently stimulated to induce detectable cellularproliferation. Activation can also be associated with induced cytokineproduction, and detectable effector functions. The term “activated Tcells” refers to, among other things, T cells that are undergoing celldivision.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which specifically binds with an antigen. Antibodies can beintact immunoglobulins derived from natural sources or from recombinantsources and can be immunoreactive portions of intact immunoglobulins.Antibodies are typically tetramers of immunoglobulin molecules. Theantibodies in the present invention may exist in a variety of formsincluding, for example, polyclonal antibodies, monoclonal antibodies,Fv, Fab and F(ab)₂, as well as single chain antibodies, humanantibodies, and humanized antibodies (Harlow et al., 1999, In: UsingAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, ColdSpring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; Bird et al., 1988, Science 242:423-426).

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limitedto, Fab, Fab′, F(ab′)₂, and Fv fragments, linear antibodies, scFvantibodies, and multispecific antibodies formed from antibody fragments.

An “antibody heavy chain,” as used herein, refers to the larger of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations.

An “antibody light chain,” as used herein, refers to the smaller of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations, κ and λ light chains refer tothe two major antibody light chain isotypes.

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

The term “antigen” or “Ag” as used herein is defined as a molecule thatprovokes an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. The skilled artisan willunderstand that any macromolecule, including virtually all proteins orpeptides, can serve as an antigen. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. A skilled artisan will understand thatany DNA, which comprises a nucleotide sequences or a partial nucleotidesequence encoding a protein that elicits an immune response thereforeencodes an “antigen” as that term is used herein. Furthermore, oneskilled in the art will understand that an antigen need not be encodedsolely by a full length nucleotide sequence of a gene. It is readilyapparent that the present invention includes, but is not limited to, theuse of partial nucleotide sequences of more than one gene and that thesenucleotide sequences are arranged in various combinations to elicit thedesired immune response. Moreover, a skilled artisan will understandthat an antigen need not be encoded by a “gene” at all. It is readilyapparent that an antigen can be generated synthesized or can be derivedfrom a biological sample. Such a biological sample can include, but isnot limited to a tissue sample, a tumor sample, a cell or a biologicalfluid.

The term “tumor antigen” as used herein refers to an antigen associatedwith a cancer cell. In some embodiments the tumor antigen is CD19, whichis associated with B cell malignancies. In some embodiments, the tumorantigen is CD30, which is typically associated with lymphomas (e.g., HL,ALCL, diffuse large cell B-lymphoma, etc. Examples of other tumorantigens include, but are not limited to, CD2, CD5, CD10, CD20, CD22,CD30, CD33, CD38, CD52, CD56, CD74, CD138, CD317, Her2, VEGFR2,EGFRviii, CXCR4, BCMA, GD2, GD3, and any other antigens over-expressedin tumor cells.

In some embodiments the tumor antigen is GD2. GD2 is adisialoganglioside that is expressed on cancer cells, such asretinoblastoma or osteosarcoma. An exemplary structure of GD2 is asfollows:

The IUPAC name for GD2 is(2R,4R,5S,6S)-2-[3-[(2S,3S,4R,6S)-6-[(2S,3R,4R,5S,6R)-5-[(2S,3R,4R,5R,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-2-[(2R,3S,4R,5R,6R)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(E)-3-hydroxy-2-(octadecanoylamino)octadec-4-enoxy]oxan-3-yl]oxy-3-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3-amino-6-carboxy-4-hydroxyoxan-2-yl]-2,3-dihydroxypropoxyl-5-amino-4-hydroxy-6-(1,2,3-trihydroxypropyl)oxane-2-carboxylicacid (see, e.g., PubChem ID 6450346).

The term “target antigen” as used herein refers to an antigen associatedwith a disease-associated target cell. Examples of target antigensinclude but are not limited to CD2, CD5, CD7, CD10, CD19, CD20, CD22,CD30, CD33, CD38, CD52, CD56, CD74, CD138, CD317, Her2, VEGFR2,EGFRviii, CXCR4, BCMA, GD2, GD3, and any other antigens over-expressedin target cells or diseased cells. In some embodiments, the diseasedcell is a B cell that produces auto-antibodies.

The term “anti-tumor effect” as used herein, refers to a biologicaleffect which can be manifested by a decrease in tumor volume, a decreasein the number of tumor cells, a decrease in the number of metastases, anincrease in life expectancy, or amelioration of various physiologicalsymptoms associated with the cancerous condition. An “anti-tumor effect”can also be manifested by the ability of the peptides, polynucleotides,cells and antibodies of the invention in prevention of the occurrence oftumor in the first place.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to which it is later to bere-introduced into the individual.

“Allogeneic” refers to a graft derived from a different animal of thesame species. “Xenogeneic” refers to a graft derived from an animal of adifferent species.

The term “cancer” as used herein is defined as disease characterized bythe rapid and uncontrolled growth of aberrant cells. Cancer cells canspread locally or through the bloodstream and lymphatic system to otherparts of the body. Examples of various cancers include, but are notlimited to, breast cancer, prostate cancer, ovarian cancer, cervicalcancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer,liver cancer, brain cancer, lymphoma, leukemia (e.g., chroniclymphocytic leukemia, acute lymphoblastic leukemia, pediatric acute Bcell leukemia, or post hematopoietic stem cell transplant relapsedleukemia), lung cancer and the like. In some embodiments, cancer refersto B-cell related malignancies (e.g., B-cell chronic lymphocyticleukemia, acute lymphoblastic leukemia, B-cell prolymphocytic leukemia,precursor B lymphoblastic leukemia, Hairy cell leukemia, diffuse large Bcell lymphoma, follicular lymphoma, marginal zone lymphoma, small celllymphocytic lymphoma, mantle cell lymphoma, Burkitt lymphoma, primarymediastinal (thymic) large B-cell lymphoma, lymphoplasmacytic lymphoma,nodal marginal zone B cell lymphoma, intravascular large B-celllymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, orplasmablastic lymphoma).

In some embodiments, cancer refers to CD30+ cancers (e.g., Hodgkin'slymphoma (HL), anaplastic large cell lymphoma (ALCL), diffuse large Bcell lymphoma, and peripheral T/NK cell lymphoma, primary effusionlymphoma, adult T-cell leukemia/lymphoma, mycosis fungoides, andextranodal natural killer/T-cell lymphoma).

In some embodiments, the cancer is a GD2-expressing cancer. In someembodiments, the cancer is retinoblastoma or osteosarcoma.Retinoblastoma is a cancer that develops from cells of the retina and isthe most common malignant tumor in children. Retinoblastoma can beidentified by the skilled practitioner, e.g., using techniques known inthe art including red reflex, Hirschberg test, an eye examination,computerized tomography (CT), magnetic resonance imaging (MRI), orultrasound. Genetic testing, e.g., mutations in the RB1 gene may also beused to identify subjects. Osteosarcoma is a tumor that occurs in thebone which is of mesenchymal origin. Osteosarcoma occurs most often inchildren and young adults. Osteosarcoma can be identified by the skilledpractitioner, e.g., using techniques known in the art including X-ray,CT scan, PET scan, bone scan, MRI, or biopsy.

The term “autoimmune disease” as used herein refers to a diseasecharacterized by an abnormal immune response of the body against thebody's own cells and tissues. The immune response may be systemic or maybe restricted to certain tissue types or organs. Examples of variousautoimmune diseases include, but are not limited to, acute disseminatedencephalomyelitis, Addison's disease, alopecia areata, autoimmunecardiomyopathy, autoimmune hemolytic anemia, autoimmune hepatitis,autoimmune lymphoproliferative syndrome, autoimmune peripheralneuropathy, autoimmune pancreatitis, autoimmune polyendocrine syndrome,autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura,autoimmune urticarial, autoimmune uveitis, Behçet's disease, Celiacdisease, Churg-Strauss syndrome, cold agglutinin disease, Crohn'sdisease, dermatomyositis, diabetes mellitus type 1, eosinophilicfasciitis, gastrointestinal pemphigoid, Goodpasture's syndrome, Graves'disease, Guillain-Barré syndrome, Hashimoto's encephalopathy,Hashimoto's thyroiditis, idiopathic thrombocytopenic purpura, lupuserythematosus, Miller-Fisher syndrome, mixed connective tissue disease,multiple sclerosis, myasthenia gravis, pemphigus vulgaris, perniciousanaemia, polymyositis, primary biliary cirrhosis, psoriasis, psoriaticarthritis, relapsing polychondritis, rheumatoid arthritis, rheumaticfever, Sjögren's syndrome, temporal arteritis, transverse myelitis,ulcerative colitis, undifferentiated connective tissue disease,vasculitis, and Wegener's granulomatosis.

The term “infection” as used herein refers to an invasion of a host'scells or tissues with an infectious organism, such as a bacteria, virus,or fungus.

An “effective amount” as used herein, means an amount which provides atherapeutic or prophylactic benefit.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide.

The term “immunoglobulin” or “Ig,” as used herein is defined as a classof proteins, which function as antibodies. Antibodies expressed by Bcells are sometimes referred to as the BCR (B cell receptor) or antigenreceptor. The five members included in this class of proteins are IgA,IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present inbody secretions, such as saliva, tears, breast milk, gastrointestinalsecretions and mucus secretions of the respiratory and genitourinarytracts. IgG is the most common circulating antibody. IgM is the mainimmunoglobulin produced in the primary immune response in most subjects.It is the most efficient immunoglobulin in agglutination, complementfixation, and other antibody responses, and is important in defenseagainst bacteria and viruses. IgD is the immunoglobulin that has noknown antibody function, but may serve as an antigen receptor. IgE isthe immunoglobulin that mediates immediate hypersensitivity by causingrelease of mediators from mast cells and basophils upon exposure toallergen.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

Unless otherwise specified, a “nucleotide sequence or nucleic acidencoding an amino acid sequence” includes all nucleotide sequences thatare degenerate versions of each other and that encode the same aminoacid sequence. The phrase nucleotide sequence that encodes a protein oran RNA may also include introns to the extent that the nucleotidesequence encoding the protein may in some version contain an intron(s).

A “lentivirus” as used herein refers to a genus of the Retroviridaefamily. Lenti viruses are unique among the retroviruses in being able toinfect non-dividing cells; they can deliver a significant amount ofgenetic information into the DNA of the host cell, so they are one ofthe most efficient methods of a gene delivery vector. HIV, SIV, and FIVare all examples of lenti viruses. Vectors derived from lenti virusesoffer the means to achieve significant levels of gene transfer in vivo.

By the term “modulating,” as used herein, is meant mediating adetectable increase or decrease in the level of a response in a subjectcompared with the level of a response in the subject in the absence of atreatment or compound, and/or compared with the level of a response inan otherwise identical but untreated subject. The term encompassesperturbing and/or affecting a native signal or response therebymediating a beneficial therapeutic response in a subject, preferably, ahuman.

“Codon-optimized” means that codons relating to a specific amino acidare optimized for translational efficiency of a gene of interest. Codonoptimization typically involves evaluating the gene or sequence ofinterest and substituting the codon with a more prevalent or commoncodon used for the same amino acid in a specific cell or species.Programs used by those in the art to evaluate codon optimization includethose provided by Integrated DNA Technologies, EnCor Biotechnology,Inc., JCat, OptimumGene™ (GenScript USA, Inc., Piscataway, N.J. 08854),etc. The sequences encoding the CAR embodiments described herein may becodon-optimized, which can increase their translational efficiency.

The term “operably linked” refers to functional linkage between aregulatory sequence and a heterologous nucleic acid sequence resultingin expression of the latter. For example, a first nucleic acid sequenceis operably linked with a second nucleic acid sequence when the firstnucleic acid sequence is placed in a functional relationship with thesecond nucleic acid sequence. For instance, a promoter is operablylinked to a coding sequence if the promoter affects the transcription orexpression of the coding sequence. Generally, operably linked DNAsequences are contiguous and, where necessary to join two protein codingregions, in the same reading frame.

The term “overexpressed” or “overexpression” is intended to indicate anabnormal level of expression (e.g., of the tumor antigen) in a cell froma disease area (e.g., a solid tumor within a specific tissue or organ ofthe patient) relative to the level of expression in a normal cell fromthat tissue or organ. Patients having solid tumors or a hematologicalmalignancy characterized by overexpression of the tumor antigen can bedetermined by standard assays known in the art.

“Parenteral” administration of an immunogenic composition includes,e.g., subcutaneous (s.c), intravenous (i.v.), intramuscular (i.m.), orintrasternal injection, or infusion techniques.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In some embodiments, the patient, subject or individual is a human. Insome embodiments, the patient, subject or individual is a child (such as18 years of age or younger).

The term “promoter” as used herein is defined as a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa polynucleotide sequence.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner. A “constitutive” promoteris a nucleotide sequence which, when operably linked with apolynucleotide which encodes or specifies a gene product, causes thegene product to be produced in a cell under most or all physiologicalconditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell substantially only whenan inducer which corresponds to the promoter is present in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide encodes or specified by a gene,causes the gene product to be produced in a cell substantially only ifthe cell is a cell of the tissue type corresponding to the promoter.

By the term “specifically binds” or “specific for”, as used herein withrespect to an antibody, is meant an antibody which recognizes a specificantigen, but does not substantially recognize or bind other molecules ina sample. For example, an antibody that specifically binds to an antigenfrom one species may also bind to that antigen from one or more species.But, such cross-species reactivity does not itself alter theclassification of an antibody as specific. In another example, anantibody that specifically binds to an antigen may also bind todifferent allelic forms of the antigen. However, such cross reactivitydoes not itself alter the classification of an antibody as specific. Insome instances, the terms “specific binding” or “specifically binding,”can be used in reference to the interaction of an antibody, a protein,or a peptide with a second chemical species, to mean that theinteraction is dependent upon the presence of a particular structure(e.g., an antigenic determinant or epitope) on the chemical species; forexample, an antibody recognizes and binds to a specific proteinstructure rather than to proteins generally. If an antibody is specificfor epitope “A”, the presence of a molecule containing epitope A (orfree, unlabeled A), in a reaction containing labeled “A” and theantibody, will reduce the amount of labeled A bound to the antibody.

The term “subject” is intended to include living organisms in which animmune response can be elicited (e.g., mammals). Examples of subjectsinclude humans, dogs, cats, mice, rats, and transgenic species thereof.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression, remission,or eradication of a disease state.

The term “therapeutically effective amount” refers to the amount of thesubject compound that will elicit the biological or medical response ofa tissue, system, or subject that is being sought by the researcher,veterinarian, medical doctor or other clinician. The term“therapeutically effective amount” includes that amount of a compoundthat, when administered, is sufficient to prevent development of, oralleviate to some extent, one or more of the signs or symptoms of thedisorder or disease being treated. The therapeutically effective amountwill vary depending on the compound, the disease and its severity andthe age, weight, etc., of the subject to be treated.

To “treat” a disease as the term is used herein, means to reduce thefrequency or severity of at least one sign or symptom of a disease ordisorder experienced by a subject.

The term “transfected” or “transformed” or “transduced” as used hereinrefers to a process by which exogenous nucleic acid is transferred orintroduced into the host cell. A “transfected” or “transformed” or“transduced” cell is one which has been transfected, transformed ortransduced with exogenous nucleic acid. The cell includes the primarysubject cell and its progeny.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,and the like.

Compositions

In some embodiments, the present invention provides a chimeric antigenreceptor (CAR) comprising (a) an extracellular domain comprising anantigen binding domain, (b) a transmembrane domain and (c) a cytoplasmicdomain. It should be appreciated that in some embodiments, CAR moleculesdescribed by the following exemplary, non-limiting arrangements are fromleft to right, N-terminus to C-terminus of the CAR. A CAR molecule asdescribed by the disclosure may comprise or further comprise any othercombination of elements as described herein.

In some embodiments, a CAR as described by the disclosure is fullyhuman. In some embodiments, a CAR has a cytoplasmic domain comprising, aIL-15Rα cytoplasmic domain in combination with one or more othercytoplasmic domains described herein, e.g., a CD3 zeta domain. In someembodiments, the arrangement of the elements of a CAR is selected fromone of the following exemplary, non-limiting arrangements:

scFv-CD28-IL-15Rα-CD3zscFv-CD28-CD27-IL-15Rα-CD3zscFv-CD28-(4-1BB)-CD27-IL-15Rα-CD3zscFv-CD8-CD27-IL-15Rα-CD3zscFv-CD8-(4-1BB)-CD27-IL-15Rα-CD3z

In some embodiments, a CAR has a cytoplasmic domain comprising, a CD27cytoplasmic domain in combination with one or more other cytoplasmicdomains described herein, e.g., a 4-1BB intracellular domain and/or aCD3 zeta domain. In some embodiments, the cytoplasmic domain furthercomprises a safety-enhancing domain, e.g., an apoptosis-inducingiCasp9-FKBP domain. In some embodiments, the arrangement of the elementsof a CAR is selected from one of the following exemplary, non-limitingarrangements:

scFv-CD28-(4-1BB)-CD27-CD3z-iCasp9-FKBPscFv-CD28-CD27-(4-1BB)-CD3z-iCasp9-FKBPscFv-CD8-(4-1BB)-CD27-CD3z-iCasp9-FKBPscFv-CD8-CD27-(4-1BB)-CD3z-iCasp9-FKBP

In some embodiments, a CAR described by the disclosure comprises anantigen binding domain specific for GD2. In some embodiments, thearrangement of the elements of the CAR is selected from one of thefollowing exemplary, non-limiting arrangements:

GD2scFv-CD28-(4-1BB)-CD27-CD3z GD2scFv-CD8-CD28-CD3zGD2scFv-CD28-(4-1BB)-CD27-CD3z-Casp9-FKBPGD2scFv-CD8-CD28-CD3z-Casp9-FKBP

Between the extracellular domain (comprising the antigen binding domain)and the transmembrane domain of the CAR, or between the cytoplasmicdomain and the transmembrane domain of the CAR, there may beincorporated a spacer or hinge domain. As used herein, the term “spacerdomain” generally means any oligo- or polypeptide that functions to linkthe transmembrane domain to the extracellular domain and/or thecytoplasmic domain in the polypeptide chain. As used herein, a hingedomain generally means any oligo- or polypeptide that functions toprovide flexibility to the CAR, or domains thereof, and/or preventsteric hindrance of the CAR, or domains thereof. In some embodiments, aspacer or hinge domain may comprise up to 300 amino acids, preferably 10to 100 amino acids and most preferably 5 to 20 amino acids. It alsoshould be appreciated that one or more spacer domains may be included inother regions of a CAR, as aspects of the disclosure are not limited inthis respect.

It is to be understood that a CAR can include a region (e.g., an antigenbinding domain, a transmembrane domain, a cytoplasmic domain, asignaling domain, a safety domain, and/or a linker, or any combinationthereof) having a sequence provided herein or a variant thereof or afragment of either one thereof (e.g., a variant and/or fragment thatretains the function required for the CAR activity) can be included in aCAR protein as described herein. In some embodiments, a variant has 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes relative to theillustrated sequence. In some embodiments, a variant has a sequence thatis at least 80%, at least 85%, at least 90%, 90%-95%, at least 95% or atleast 99% identical to the illustrated sequence. In some embodiments, afragment is 1-5, 5-10, 10-20, 20-30, 30-40, or 40-50 amino acids shorterthan a sequence provided herein. In some embodiments, a fragment isshorter at the N-terminal, C-terminal, or both terminal regions of thesequence provided. In some embodiments, a fragment contains 80%-85%,85%-90%, 90%-95%, or 95%-99% of the number of amino acids in a sequenceprovided herein.

In some embodiments, the spacer and/or hinge sequences of the CAR areselected from one or more of the following exemplary sequences:

Spacer Sequences: (SEQ ID NO: 1) GGGGS (SEQ ID NO: 2) GGGGSGGGGS(SEQ ID NO: 3) GGGGS × 3 (SEQ ID NO: 4) GS18: GSTSGGGSGGGSGGGGSS(SEQ ID NO: 5) 218S: GSTSGSGKPGSSEGSTKG (SEQ ID NO: 6) GS8: GGGGSGGGHinge Sequences: (SEQ ID NO: 7) Native: VEPKSCDKTHTCPPCP (SEQ ID NO: 8)C233S: LDPKSSDKTHTCPPCP (SEQ ID NO: 9) C233P: VEPKSPDKTHTCPPCP(SEQ ID NO: 10) Delta5: LDKTHTCPPCP

Antigen Binding Domains

In some embodiments, the CAR of the invention comprises an antigenbinding domain. The choice of binding domain depends upon the type andnumber of ligands that define the surface of a target cell. For example,the antigen binding domain may be chosen to recognize a ligand that actsas a cell surface marker on target cells associated with a particulardisease state, such as cancer or an autoimmune disease. Thus examples ofcell surface markers that may act as ligands for the antigen bindingdomain in the CAR of the invention include those associated with cancercells and other forms of diseased cells, for example, autoimmune diseasecells and pathogen infected cells. In some embodiments, the CAR of theinvention is engineered to target a tumor antigen of interest by way ofengineering a desired antigen binding domain that specifically binds toan antigen on a tumor cell. In the context of the present invention,“tumor antigen” refers to antigens that are common to specifichyperproliferative disorders such as cancer. The antigens discussedherein are merely included by way of example. The list is not intendedto be exclusive and further examples will be readily apparent to thoseof skill in the art.

The antigen binding domain of the CAR may target, for example, CD19,CD30, or GD2. Other examples of target antigens include, but are notlimited, to CD2, CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD38,CD52, CD56, CD74, CD138, CD317, Her2, VEGFR2, EGFRviii, CXCR4, BCMA,GD2, GD3, and any other antigens over-expressed in target or diseasedcells. Other antigens specific for cancer that may be targeted at taughtin PCT publication No. WO2013/123061 (page 20), which is incorporatedherein by reference with respect to the antigens recited therein.

The antigen binding domain can be any domain that binds to the antigenincluding but not limited to monoclonal antibodies, scFvs, polyclonalantibodies, synthetic antibodies, human antibodies, humanizedantibodies, and fragments thereof. In some instances, it is beneficialfor the antigen binding domain to be derived from the same species inwhich the CAR will ultimately be used in. For example, for use inhumans, it may be beneficial for the antigen binding domain of the CARto comprise a human antibody or fragment thereof. Thus, in someembodiments, the antigen binding domain comprises a human antibody or afragment thereof.

An antigen binding domain (e.g., an scFV) that “specifically binds” to atarget or an epitope is a term understood in the art, and methods todetermine such specific binding are also known in the art. A molecule issaid to exhibit “specific binding” if it reacts or associates morefrequently, more rapidly, with greater duration and/or with greateraffinity with a particular target antigen than it does with alternativetargets. An antibody “specifically binds” to a target antigen if itbinds with greater affinity, avidity, more readily, and/or with greaterduration than it binds to other substances. For example, an antigenbinding domain (e.g., an scFV) that specifically binds to GD2 or anepitope therein is an antibody that binds this target antigen withgreater affinity, avidity, more readily, and/or with greater durationthan it binds to other antigens or other epitopes in the same antigen.It is also understood by reading this definition that, for example, anantigen binding domain (e.g., an scFV) that specifically binds to afirst target antigen may or may not specifically bind to a second targetantigen. As such, “specific binding” does not necessarily require(although it can include) exclusive binding. Generally, but notnecessarily, reference to binding means specific binding. In someembodiments, antigen binding domains (e.g., scFVs) described herein havea suitable binding affinity to GD2. As used herein, “binding affinity”refers to the apparent association constant or K_(A). The K_(A) is thereciprocal of the dissociation constant (K_(D)). The antibody describedherein may have a binding affinity (K_(A)) of at least 10⁵, 10⁶, 10⁷,10⁸, 10⁹, 10¹⁰ M, or higher. An increased binding affinity correspondsto a decreased K_(D). Higher affinity binding of an antibody to a firsttarget relative to a second target can be indicated by a higher K_(A)(or a smaller numerical value K_(D)) for binding the first target thanthe K_(A) (or numerical value K_(D)) for binding the second target. Insuch cases, the antibody has specificity for the first target relativeto the second target. Differences in binding affinity (e.g., forspecificity or other comparisons) can be at least 1.5, 2, 3, 4, 5, 10,15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000 or 10⁵ fold.

Binding affinity can be determined by a variety of methods includingequilibrium dialysis, equilibrium binding, gel filtration, ELISA,surface plasmon resonance, or spectroscopy (e.g., using a fluorescenceassay). Exemplary conditions for evaluating binding affinity are in,e.g., TRIS-buffer (50 mM TRIS, 150 mM NaCl, 5 mM CaCl2 at pH7.5). Thesetechniques can be used to measure the concentration of bound bindingprotein as a function of target protein concentration. The concentrationof bound binding protein ([Bound]) is related to the concentration offree target protein ([Free]) and the concentration of binding sites forthe binding protein on the target where (N) is the number of bindingsites per target molecule by the following equation:

[Bound]=[N][Free]/(Kd+[Free])

It is not always necessary to make an exact determination of K_(A),though, since sometimes it is sufficient to obtain a quantitativemeasurement of affinity, e.g., determined using a method such as ELISAor FACS analysis, is proportional to K_(A), and thus can be used forcomparisons, such as determining whether a higher affinity is, e.g.,2-fold higher, to obtain a qualitative measurement of affinity, or toobtain an inference of affinity, e.g., by activity in a functionalassay, e.g., an in vitro or in vivo assay.

For in vivo use of antibodies in humans, it may be preferable to usehuman antibodies. Completely human antibodies are particularly desirablefor therapeutic treatment of human subjects. Human antibodies can bemade by a variety of methods known in the art including phage displaymethods using antibody libraries derived from human immunoglobulinsequences, including improvements to these techniques. See, also, U.S.Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO98/50433, WO 98/24893, WO98/16654, WO 96/34096, WO 96/33735, andWO91/10741; each of which is incorporated herein by reference in itsentirety. A human antibody can also be an antibody wherein the heavy andlight chains are encoded by a nucleotide sequence derived from one ormore sources of human DNA. Human antibodies can also be produced usingtransgenic mice which are incapable of expressing functional endogenousimmunoglobulins, but which can express human immunoglobulin genes. Forexample, the human heavy and light chain immunoglobulin gene complexesmay be introduced randomly or by homologous recombination into mouseembryonic stem cells. Alternatively, the human variable region, constantregion, and diversity region may be introduced into mouse embryonic stemcells in addition to the human heavy and light chain genes. The mouseheavy and light chain immunoglobulin genes may be renderednon-functional separately or simultaneously with the introduction ofhuman immunoglobulin loci by homologous recombination. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region (JH) gene in chimeric and germ-line mutant miceresults in complete inhibition of endogenous antibody production. Themodified embryonic stem cells are expanded and microinjected intoblastocysts to produce chimeric mice. The chimeric mice are then bred toproduce homozygous offspring which express human antibodies. Thetransgenic mice are immunized in the normal fashion with a selectedantigen, e.g., all or a portion of a polypeptide of the invention.

Antibodies directed against an antigen can be obtained from theimmunized, transgenic mice using conventional hybridoma technology. Thehuman immunoglobulin transgenes harbored by the transgenic micerearrange during B cell differentiation, and subsequently undergo classswitching and somatic mutation. Thus, using such a technique, it ispossible to produce therapeutically useful IgG, IgA, IgM and IgEantibodies, including, but not limited to, IgG1 (gamma 1) and IgG3. Fora detailed discussion of this technology for producing human antibodiesand human monoclonal antibodies and protocols for producing suchantibodies, see, e.g., PCT Publication Nos. WO2014/055771, WO 98/24893,WO 96/34096, and WO 96/33735; and U.S. Pat. Nos. 5,413,923; 5,625,126;5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; and 5,939,598,each of which is incorporated by reference herein in their entirety.

A “humanized” antibody retains a similar antigenic specificity as theoriginal antibody, i.e., in the present invention, the ability to bindan antigen described herein, for example, CD19, CD30, or GD2.

In some embodiments, the antigen binding domain of the CAR of theinvention targets CD19. In some embodiments, the antigen binding moietyportion in the CAR of the invention is a fully human anti-CD19 scFV. Insome embodiments, the anti-CD19 scFV comprises the sequence below, orthe complementarity determining regions (CDRs, underlined below andnumbered CDR1-3) contained within the sequence below.

Exemplary CD19 scFv: Light chain: (SEQ ID NO: 11)DIQMTQTTSSLSASLGDRVTISC RASQDISKYLN(CDR1) WYQQKPDGTVKLLIY HTSRLHS(CDR2)GVPSRFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPYT(CDR3) FGGGTKLEIT Heavy chain:(SEQ ID NO: 12) EVKLQESGPGLVAPSQSLSVTCTVSGVSLP DYGVS(CDR1)WIRQPPRKGLEWLG VIWGSETTY(CDR2)  YNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDY(CDR3) WGQGTSVTVSS

In some embodiments, the antigen binding domain of the CAR of thedisclosure targets CD30. In some embodiments, the antigen binding moietyportion in the CAR of the disclosure is a fully human anti-CD30 scFV. Insome embodiments, the anti-CD30 scFV comprises a sequence below, or thecomplementarity determining regions (CDRs, underlined below and numberedCDR1-3) contained within the sequences below:

Exemplary CD30 scFvs: AC10 scFv Light chain: (SEQ ID NO: 13)DIVMTQSPDSLAVSLGERATINC KASQSVDFDGDSYMN(CDR1)WYQQKPGQPPKLLIY AASNLES(CDR2)GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSNEDPWT(CDR3) FGQGTKVEIKAC10 scFv Heavy chain: (SEQ ID NO: 14)VHQIQLVQSGAEVKKPGASVKVSCKAS GYTFTDYYIT(CDR1)WVRQAPGQGLEWMG WIYPGSGNTKYNEKFKG(CDR2)RVTMTRDTSISTAYMELSRLRSDDTAVYYCANYGNYWFAY(CDR3) WGQGTLVTVSS5F11 scFv Light Chain: (SEQ ID NO: 15)DIQMTQSPTSLSASVGDRVTITC RASQGISSWLT(CDR1) WYQQKPEKAPKSLIY AASSLQS(CDR2)GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDSYPIT(CDR3) FGQGTRLEIK5F11 scFv Heavy Chain: (SEQ ID NO: 16)QVQLQQWGAGLLKPSETLSLTCAVYGGSFS AYYWS(CDR1)WIRQPPGKGLEWIG DINHGGGTNYNPSLKS(CDR2)RVTISVDTSKNQFSLKLNSVTAADTAVYYCASLTAY(CDR3) WGQGSLVTVSS

In some embodiments, the antigen binding domain of the CAR of theinvention is specific for GD2. In some embodiments, the antigen bindingmoiety portion in the CAR of the invention is an anti-GD2 scFV, such asa fully human anti-GD2 scFV. In some embodiments, the anti-GD2 scFVcomprises the sequence(s) of the light and/or heavy chain variableregions of hu3F8, c.60C3 or hu14.18, or the complementarity determiningregions (CDRs) contained within the light and/or heavy chain variableregions of hu3F8, c.60C3 or hu14.18 (see, e.g., Yu, A. L., et al.,Anti-GD2 antibody with GM-CSF, interleukin-2, and isotretinoin forneuroblastoma. N Engl J Med, 2010. 363(14): p. 1324-34; Ahmed, M. and N.K. Cheung, Engineering anti-GD2 monoclonal antibodies for cancerimmunotherapy. FEBS Lett, 2014. 588(2): p. 288-97; and Alvarez-Rueda,N., et al., Binding activities and antitumor properties of a newmouse/human chimeric antibody specific for GD2 ganglioside antigen. ClinCancer Res, 2007. 13(18 Pt 2): p. 5613s-5620s). In some embodiments, theanti-GD2 scFV comprises the variable heavy chain (VH) and variable lightchain (VL) sequences below, or the complementarity determining regions(CDRs, underlined below) contained within the sequences below.

Exemplary GD2 scFV:

VH: (CDR regions underlined) (SEQ ID NO: 17)QVQLVESGPGVVQPGRSLRISCAVSGFSVT NYGVH WVRQPPGKGLEWLG VIWAGGITNYNSAFMSRLTISKDNSKNTVYLQMNSLRAEDTAMYYCAS RGGHYGYALDY WGQGTLVTVSSVL: (CDR regions underlined) (SEQ ID NO: 18)EIVMTQTPATLSVSAGERVTITC KASQSVSNDVT WYQQKPGQAPRLLIY SASNRYSGVPARFSGSGYGTEFTFTISSVQSEDFAVYFC QQDYSS   FGQGTKLEIKOther exemplary_anti-GD2 scFV sequencesare shown below with an exemplary 218S linker sequence underlined.hu3F8 scFv: (VH and VL linked by 218S linker) (SEQ ID NO: 19)QVQLVESGPGVVQPGRSLRISCAVSGFSVTNYGVHWVRQPPGKGLEWLGVIWAGGITNYNSAFMSRLTISKDNSKNTVYLQMNSLRAEDTAMYYCASRGGHYGYALDYWGQGTLVTVSSGSTSGSGKPGSSEGSTKGEIVMTQTPATLSVSAGERVTITCKASQSVSNDVTWYQQKPGQAPRLLIYSASNRYSGVPARFSGSGYGTEFTFTISSVQSEDFAVYFC QQDYSSFGQGTKLEIKC60c3 ScFv: (VH and VL linked by 218S linker) (SEQ ID NO: 20)EVKLVESGGGLVLPGDSLRLSCATSEFTFTDYYMTWVRQPPRKALEWLGFIRNRANGYTTEYNPSVKGRFTISRDNSQSILYLQMNTLRTEDSATYYCARVSNWAFDYWGQGTTLTVSSGSTSGSGKPGSSEGSTKGDVVMTQTPLSLPVSLGDQASISCRSSQSLLKNNGNTFLHWYLQKSGQSPKLLIYKVSNRLSGVPDRFSGSGSGTYFTLKISRVEAEDL GVYFCSQSTHIPYTFGGGTKLEIKHu14.18 scFv: : (VH and VL linked by 218S linker) (SEQ ID NO: 21)EVQLVQSGAEVEKPGASVKISCKASGSSFTGYNMNWVRQNIGKSLWIGAIDPYYGGTSYNQKFKFRATLTVDKSTSTAYMHLKSLRSEDTAVYYCVSGMEYWGQGTSVTVSSGSTSGSGKPGSSEGSTKGDVVMTQTPLSLPVTPGEPASISCRSSQSLVHRNGNTYLHWYLQKPGQSKPLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELK

Further Extracellular Domain

In some embodiments, the CAR is designed to include an extracellular Tcell co-stimulatory domain such as CD28 extracellular domain, or aportion thereof. The extracellular domain may serve as a hinge domain orT cell activation domain. Examples include the CD28 extracellulardomain, which has 50 amino acids. An exemplary sequence of the CD28extracellular domain is:

(SEQ ID NO: 22) YVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSK P.

Transmembrane Domain

With respect to the transmembrane domain, the CAR can be designed tocomprise a transmembrane domain that is fused to the extracellulardomain (e.g., the antigen binding domain) of the CAR. Any transmembranedomain is contemplated for use herein as long as the domain is capableof anchoring a CAR comprising the domain to a cell membrane. In someembodiments, the transmembrane domain that naturally is associated withone of the domains in the CAR is used. In some instances, thetransmembrane domain can be selected or modified by amino acidsubstitution to avoid binding of such domains to the transmembranedomains of the same or different surface membrane proteins to minimizeinteractions with other members of the receptor complex. One skilled inthe art would appreciate that the full transmembrane domain, or portionthereof, is implemented with the cytoplasmic domain, or a portionthereof. Typically, the transmembrane and cytoplasmic domains used wouldbe contiguous portions of the CD28 sequence.

The transmembrane domain may be derived either from a natural or from asynthetic source. Where the source is natural, the domain may be derivedfrom any membrane-bound or transmembrane protein. Transmembrane domainsof particular use in this invention may be derived from (e.g., compriseat least the transmembrane domain(s) of) the alpha, beta or zeta chainof the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9,CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.Transmembrane domains can be identified using any method known in theart or described herein, e.g., by using the UniProt Database.

In some embodiments, the transmembrane domain may be synthetic, in whichcase it will comprise predominantly hydrophobic residues such as leucineand valine. Preferably a triplet of phenylalanine, tryptophan and valinewill be found at each end of a synthetic transmembrane domain.Optionally, a short oligo- or polypeptide linker, preferably between 2and 10 amino acids in length may form the linkage between thetransmembrane domain and the cytoplasmic signaling domain of the CAR. Aglycine-serine doublet provides a particularly suitable linker.

In some embodiments, the transmembrane domain in the CAR of theinvention is the CD8 transmembrane domain. Sequences of CD8 for thispurposes are taught in PCT pub no. WO2014/055771.

In some embodiments, the transmembrane domain in the CAR of theinvention is a CD28 transmembrane domain. An exemplary sequence of CD28is provided below, as well as an exemplary transmembrane domainsequence. In some embodiments, the CD28 transmembrane domain comprisesthe exemplary transmembrane domain sequence below, or a fragment orvariant thereof that is capable of anchoring a CAR comprising thesequence to a cell membrane.

CD28 (amino acids 19-220) (SEQ ID NO: 23)NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAY RSCD28 (amino acids 153-179, transmembrane domain) (SEQ ID NO: 24)FWVLVVVGGVLACYSLLVTVAFIIFWV

In some embodiments, the CAR of the invention is comprises a region ofCD28 that contains all or part of an extracellular domain, all or partof a transmembrane domain and all or part of a cytoplasmic domain. Anexemplary sequence of a region of CD28 for inclusion in a CAR isprovided below. In some embodiments, the CD28 transmembrane domaincomprises the exemplary transmembrane domain sequence below, or afragment or variant thereof that is capable of anchoring a CARcomprising the sequence to a cell membrane.

CD28 region (SEQ ID NO: 25)IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPR DFAAYRSAS

In some embodiments, the transmembrane domain of the CAR of theinvention comprises a hinge domain such as a CD8 hinge domain. Anexemplary CD8 hinge domain sequence is provided below. In someembodiments, the CD8 hinge domain comprises the exemplary sequencebelow, or a fragment or variant thereof that is capable of providingflexibility to or preventing steric hindrance of the CAR or thedomain(s) attached to the hinge domain. In some instances, a variety ofhuman hinges can be employed as well including the human Ig(immunoglobulin) hinge.

CD8 hinge domain (SEQ ID NO: 26)AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

Cytoplasmic Domain

In some embodiments, the cytoplasmic domain or otherwise theintracellular signaling domain of the CAR of the invention isresponsible for activation of at least one of the normal effectorfunctions of the immune cell in which the CAR has been placed in. Theterm “effector function” refers to a specialized function of a cell.Effector function of a T cell, for example, may be cytolytic activity orhelper activity including the secretion of cytokines. Thus the term“intracellular signaling domain” refers to the portion of a proteinwhich transduces the effector function signal and directs the cell toperform a specialized function. While usually the entire intracellularsignaling domain can be employed, in many cases it is not necessary touse the entire domain. To the extent that a truncated portion of theintracellular signaling domain is used, such truncated portion may beused in place of the intact domain as long as it transduces the effectorfunction signal. The term intracellular signaling domain is thus meantto include any truncated portion of the intracellular signaling domainsufficient to transduce the effector function signal.

In some embodiments, the cytoplasmic domain comprises an IL-15Rαcytoplasmic domain. In some embodiments, the intracellular IL-15Rαcytoplasmic domain displays effector signaling function that enhancesimmune effector activities including, but not limited to cellproliferation and cytokine production. An exemplary IL-15Rα cytoplasmicdomain sequence is provided below. In some embodiments, the IL-15Rαcytoplasmic domain comprises the exemplary sequence below, or a fragmentor variant thereof that, when included in a CAR, has the same or animproved function (such as cytolytic activity, cell proliferation orsecretion of cytokines) compared to a CAR comprising the exemplarysequence below. The function may be tested using a method providedherein, such as the method provided in Example 1.

IL- 15R intracellular domain (SEQ ID NO: 27)KSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL

In some embodiments, the cytoplasmic domain comprises a CD27intracellular domain (e.g., CD27 cytoplasmic domain). In someembodiments, the intracellular CD27 cytoplasmic domain displays effectorsignaling function that enhances immune effector activities including,but not limited to cell proliferation and cytokine production. Anexemplary CD27 cytoplasmic domain sequence is provided below. In someembodiments, the CD27 cytoplasmic domain comprises the exemplarysequence below, or a fragment or variant thereof that, when included ina CAR, has the same or an improved function (such as cytolytic activity,cell proliferation or secretion of cytokines) compared to a CARcomprising the exemplary sequence below. The function may be testedusing any suitable method known in the art.

CD27 intracellular domain (SEQ ID NO: 38)QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP

Examples of other intracellular signaling domains for use in the CAR ofthe invention include the cytoplasmic sequences of the T cell receptor(TCR) and co-receptors that act in concert to initiate signaltransduction following antigen receptor engagement, as well as anyfragment or variant of these sequences and any synthetic sequence thathas the same functional capability.

It is known that signals generated through the endogenous TCR alone areinsufficient for full activation of the T cell and that a secondary orco-stimulatory signal is also required. Thus, T cell activation can bemediated by two distinct classes of cytoplasmic signaling sequences:those that initiate antigen-dependent primary activation through the TCR(primary cytoplasmic signaling sequences) and those that act in anantigen-independent manner to provide a secondary or co-stimulatorysignal (secondary cytoplasmic signaling sequences).

Primary cytoplasmic signaling sequences regulate primary activation ofthe TCR complex either in a stimulatory way, or in an inhibitory way.Primary cytoplasmic signaling sequences that act in a stimulatory mannermay contain signaling motifs which are known as immunoreceptortyrosine-based activation motifs or ITAMs. Examples of ITAM containingprimary cytoplasmic signaling sequences that are of particular use inthe invention include those derived from TCR zeta, FcR gamma, FcR beta,CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.It is particularly preferred that cytoplasmic signaling molecule in theCAR of the invention comprises a cytoplasmic signaling sequence derivedfrom CD3zeta. Exemplary CD3 zeta domain sequences are provided below. Insome embodiments, the CD3zeta signaling domain comprises one of theexemplary sequences below, or a fragment or variant thereof that, whenincluded in a CAR, has the same or an improved function (such ascytolytic activity or secretion of cytokines) compared to a CARcomprising the exemplary sequence below. The function may be testedusing a method provided herein, such as the method provided in Example1.

CD3 zeta signaling domain (SEQ ID NO: 39)RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPR CD3 zeta signaling domain (SEQ ID NO: 40)RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPR 

The cytoplasmic domain of the CAR can be designed to comprise a CD3-zetasignaling domain combined with any other desired cytoplasmic domain(s)useful in the context of the CAR of the invention. For example, thecytoplasmic domain of the CAR can comprise a CD3 zeta domain and acostimulatory signaling region. The costimulatory signaling regionrefers to a portion of the CAR comprising the intracellular domain of acostimulatory molecule. Thus, while the invention in exemplifiedprimarily with 4-1BB, CD28, and CD27 as the co-stimulatory signalingelement, other additional costimulatory elements are within the scope ofthe invention. Exemplary co-stimulatory signaling regions include 4-1BB,CD21, CD28, CD27, CD127, ICOS, IL-15Rα, and OX40.

In some embodiments, the cytoplasmic domain of a CAR can be designed tocomprise an IL-15Rα cytoplasmic domain and a CD3-zeta signaling domaincombined with any other desired cytoplasmic domain(s) useful in thecontext of the CAR of the invention. For example, the cytoplasmic domainof the CAR can comprise an IL-15Rα cytoplasmic domain, a CD3 zeta domainand a costimulatory signaling region. The costimulatory signaling regionrefers to a portion of the CAR comprising the intracellular domain of acostimulatory molecule. Thus, while the invention in exemplifiedprimarily with 4-1BB, CD28, CD137 and CD27 as the co-stimulatorysignaling element, other additional costimulatory elements are withinthe scope of the invention.

The cytoplasmic domain of the CAR can be designed to comprise CD27cytoplasmic domain and a CD3-zeta signaling domain combined with anyother desired cytoplasmic domain(s) useful in the context of the CAR ofthe disclosure. For example, the cytoplasmic domain of the CAR cancomprise CD27 cytoplasmic domain, a CD3 zeta domain and a costimulatorysignaling region. The costimulatory signaling region refers to a portionof the CAR comprising the intracellular domain of a costimulatorymolecule. Thus, while the disclosure is exemplified primarily withIL-15Rα, 4-1BB, CD28, CD137 and CD27 as the co-stimulatory signalingelement, other additional costimulatory elements are within the scope ofthe disclosure. Example sequences of co-stimulatory signaling regionsare shown below.

CD28 (amino acids 180-220, cytoplasmic domain) (SEQ ID NO: 41)RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS4-1BB (CD137) intracellular TRAF binding domain (SEQ ID NO: 42)KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL ICOS intracellular domain(SEQ ID NO: 43) CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLOX40 intracellular domain (SEQ ID NO: 44)ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI CD27 intracellular domain(SEQ ID NO: 38) QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSPCD127 intracellular domain (SEQ ID NO: 45)KRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQ NQIL-15Ra intracellular domain (SEQ ID NO: 27)KSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL

In some embodiments, CAR of the invention comprises the apoptosisinducing gene Casp9 or a domain or truncated version thereof. Anexemplary Casp9 sequence and truncated sequence is below. In someembodiments, the CAR comprises a 2A peptide linker between a CD3 zetadomain and Casp9.

CASP9 amino acid sequence (SEQ ID NO: 28)MDEADRRLLRRCRLRLVEELQVDQLWDALLSRELFRPHMIEDIQRAGSGSRRDQARQLIIDLETRGSQALPLFISCLEDTGQDMLASFLRTNRQAAKLSKPTLENLTPVVLRPEIRKPEVLRPETPRPVDIGSGGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS A truncated CASP9 amino acid sequence(SEQ ID NO: 29) VGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS

In some embodiments, the CAR further comprises a mutated FK506 bindingprotein (e.g., FKBPf36v) motif. An exemplary mutated FK506 bindingprotein motif is provided below.

FKBP f36v amino acid sequence (SEQ ID NO: 30)MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVF DVELLKLE

The cytoplasmic signaling sequences within the cytoplasmic signalingportion of the CAR of the invention may be linked to each other in arandom or specified order. Optionally, a short oligo- or polypeptidelinker or spacer, preferably between 5 and 20 amino acids in length maybe inserted between cytoplasmic domains. A GGGGS (SEQ ID NO: 1) or(GGGGS)×3 (SEQ ID NO: 3) provides a particularly suitable linker.

In some embodiments, a CAR comprises or consists of the sequence below,which is broken down by exemplary domains included therein (domain namesappear in bold after each domain in the exemplary CARs):

(CD19 scFv domain) DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS AA(CD28 transmembrane/cytoplasmic domain)IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAP PRDFAAYRSAS (Linker)GGGGSGGGGS (IL-15Ralpha cytoplasmic signal domain)KSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL  (Linker) GGGGSGGGGS (CD3zeta signal domain)  (SEQ ID NO: 31)RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPR 

In some embodiments, a CAR comprises or consists of the sequence below,which is broken down by exemplary domains included therein (domain namesappear in bold after each domain in the exemplary CARs):

(CD30 scFv domain) QVQLQQWGAGLLKPSETLSLTCAVYGGSFSAYYWSWIRQPPGKGLEWIGDINHGGGTNYNPSLKSRVTISVDTSKNQFSLKLNSVTAADTAVYYCASLTA YWGQGSLVTVSS (Linker)GSTSGSGKPGSSEGSTKG  (CD30 scFv domain)DIQMTQSPTSLSASVGDRVTITCRASQGISSWLTWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDSYPITFGQ GTRLEIK (Linker)GSTSGSGKPGSSEGSTKG  (CD 28 transmembrane domain)FWVLVVVGGVLACYSLLVTVAFIIFWV  (4-1BB intracellular domain)KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL  (CD27 intracellular domain)QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP  (Linker)GSTSGSGKPGSSEGSTKG  (CD3 zeta domain)RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR (Linker) GSTSGSGKPGSSEGSTKG  (truncated iCasp9 domain)VGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (FKBP domain) (SEQ ID NO: 32)MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATL VFDVELLKLE

In some embodiments, the above exemplary, non-limiting arrangements arefrom left to right, N-terminus to C-terminus of the CAR. The CAR maycomprise or further comprise any other combination of elements asdescribed herein.

In some embodiments, a CAR comprises or consists of one the exemplarysequences below, which is broken down by exemplary domains includedtherein (domain names appear in bold after each domain in the exemplaryCARs):

CAR 1  (GD2 scFv heavy chain)QVQLVESGPGVVQPGRSLRISCAVSGFSVTNYGVHWVRQPPGKGLEWLGVIWAGGITNYNSAFMSRLTISKDNSKNTVYLQMNSLRAEDTAMYYCASRGGHYGYALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K (218S Linker) GSTSGSGKPGSSEGSTKG  (GD2 scFv light chain)EIVMTQTPATLSVSAGERVTITCKASQSVSNDVTWYQQKPGQAPRLLIYSASNRYSGVPARFSGSGYGTEFTFTISSVQSEDFAVYFCQQDYSSFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (CD28 transmembrane domain)GSTSGSGKPGSSEGSTKGFWVLVVVGGVLACYSLLVTVAFIIFWV (4-1BB intracellular domain) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (CD27 intracellular domain)QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP  (218S Linker)GSTSGSGKPGSSEGSTKG  (CD3zeta)RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP  PR (218S Linker) GSTSGSGKPGSSEGSTKG  (truncated Casp9 domain)VGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSL HFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYG TDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSN PEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDI FEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS  (FKBP domain)(SEQ ID NO: 33) MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQE VIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE  CAR 2  (VH hu3F8)QVQLVESGPGVVQPGRSLRISCAVSGFSVTNYGVHWVRQPPGKGLEWLGVIWAGGI TNYNSAFMSRLTISKDNSKNTVYLQMNSLRAEDTAMYYCASRGGHYGYALDYWG  QGTLVTVSS (218S Linker) GSTSGSGKPGSSEGSTKG  (VL hu3F8)EIVMTQTPATLSVSAGERVTITCKASQSVSNDVTWYQQKPGQAPRLLIYSASNRYSG VPARFSGSGYGTEFTFTISSVQSEDFAVYFCQQDYSSFGQGTKLEIK (CD28 transmembrane domain) FWVLVVVGGVLACYSLLVTVAFIIFWV (4-1BB intracellular domain) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (CD27 intracellular domain)QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP  (218S Linker)GSTSGSGKPGSSEGSTKG  (CD3zeta)RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP  PR (218S Linker) GSTSGSGKPGSSEGSTKG  (truncated Casp9)VGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSL HFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYG TDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSN PEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDI FEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS  (FKBP domain)(SEQ ID NO: 34) MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQE VIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE  CAR 3  (VH C60c3)EVKLVESGGGLVLPGDSLRLSCATSEFTFTDYYMTWVRQPPRKALEWLGFIRNRAN GYTTEYNPSVKGRFTISRDNSQSILYLQMNTLRTEDSATYYCARVSNWAFDYWGQG  TTLTVSS (218S Linker) GSTSGSGKPGSSEGSTKG  (VL C60c3)DVVMTQTPLSLPVSLGDQASISCRSSQSLLKNNGNTFLHWYLQKSGQSPKLLIYKVS NRLSGVPDRFSGSGSGTYFTLKISRVEAEDLGVYFCSQSTHIPYTFGGGTKLEIK (CD 28 transmembrane domain) FWVLVVVGGVLACYSLLVTVAFIIFWV (4-1BB intracellular domain) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (CD27 intracellular domain)QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP  (218S Linker)GSTSGSGKPGSSEGSTKG  (CD3zeta)RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP  PR (218S Linker) GSTSGSGKPGSSEGSTKG  (truncated Casp9)VGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSL HFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYG TDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSN PEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDI FEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS  (FKBP domain)(SEQ ID NO: 35) MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQE VIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE  CAR 4 (VH Hu14.18) EVQLVQSGAEVEKPGASVKISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYG GTSYNQKFKGRATLTVDKSTSTAYMHLKSLRSEDTAVYYCVSGMEYWGQGTSVTV  SS (218S Linker) GSTSGSGKPGSSEGSTKG  (VL Hu14.18)DVVMTQTPLSLPVTPGEPASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVS NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELK (CD28 transmembrane domain) FWVLVVVGGVLACYSLLVTVAFIIFWV (4-1BB intracellular domain) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (CD27 intracellular domain)QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP  (218S Linker)GSTSGSGKPGSSEGSTKG  (CD3zeta)RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP  PR (218S Linker) GSTSGSGKPGSSEGSTKG  (truncated Casp9)VGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSL HFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYG TDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSN PEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDI FEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS  (FKBP domain)(SEQ ID NO: 36) MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQE VIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE 

In some embodiments, the above exemplary, non-limiting arrangements arefrom left to right, N-terminus to C-terminus of the CAR. The CAR maycomprise or further comprise any other combination of elements asdescribed herein.

Vectors

In some embodiments, the present invention encompasses a DNA constructcomprising sequences of a CAR, wherein the sequence comprises thenucleic acid sequence of an antigen binding domain operably linked tothe nucleic acid sequence of transmembrane domain and a cytoplasmicdomain. An exemplary cytoplasmic domain that can be used in a CAR of theinvention includes but is not limited to the cytoplasmic domain ofIL-15Rα and the signaling domain of CD3-zeta. In some embodiments, a CARcomprises the intracellular domain of CD28, 4-1BB, and/or CD27 and thesignaling domain of CD3-zeta. In some instances, a CAR can furthercomprise the apoptosis inducing gene Casp9.

In some embodiments, the arrangement of the elements of the CAR isselected from one of the following exemplary, non-limiting arrangements:

scFv-CD28-IL-15Rα-CD3zscFv-CD28-CD27-IL-15Rα-CD3zscFv-CD28-(4-1BB)-CD27-IL-15Rα-CD3zscFv-CD8-CD27-IL-15Rα-CD3zscFv-CD8-(4-1BB)-CD27-IL-15Rα-CD3zscFv-CD28-(4-1BB)-CD27-CD3z-iCasp9-FKBPscFv-CD28-CD27-(4-1BB)-CD3z-iCasp9-FKBPscFv-CD8-(4-1BB)-CD27-CD3z-iCasp9-FKBPscFv-CD8-CD27-(4-1BB)-CD3z-iCasp9-FKBP

GD2scFv-CD28-(4-1BB)-CD27-CD3z GD2scFv-CD8-CD28-CD3zGD2scFv-CD28-(4-1BB)-CD27-CD3z-Casp9-FKBPGD2scFv-CD8-CD28-CD3z-Casp9-FKBP

In some embodiments, the above exemplary, non-limiting arrangements arefrom left to right, N-terminus to C-terminus of the CAR. The CAR maycomprise or further comprise any other combination of elements asdescribed herein.

The nucleic acid sequences coding for the desired molecules can beobtained using recombinant methods known in the art, such as, forexample by screening libraries from cells expressing the gene, byderiving the gene from a vector known to include the same, or byisolating directly from cells and tissues containing the same, usingstandard techniques. Alternatively, the gene of interest can be producedsynthetically, rather than cloned.

The present invention also provides vectors in which a DNA of thepresent invention is inserted. Vectors derived from retroviruses such asthe lentivirus are suitable tools to achieve long-term gene transfersince they allow long-term, stable integration of a transgene and itspropagation in daughter cells. Lenti viral vectors have the addedadvantage over vectors derived from onco-retroviruses such as murineleukemia viruses in that they can transduce non-proliferating cells,such as hepatocytes. They also have the added advantage of lowimmunogenicity. In another embodiment, the desired CAR can be expressedin the cells by way of transposons.

In brief summary, the expression of natural or synthetic nucleic acidsencoding CARs is typically achieved by operably linking a nucleic acidencoding the CAR polypeptide or portions thereof to a promoter, andincorporating the construct into an expression vector. The vectors canbe suitable for replication and integration into eukaryotes. Typicalcloning vectors contain transcription and translation terminators,initiation sequences, and promoters useful for regulation of theexpression of the desired nucleic acid sequence. The expressionconstructs of the present invention may also be used for nucleic acidimmunization and gene therapy, using standard gene delivery protocols.Methods for gene delivery are known in the art. See, e.g., U.S. Pat.Nos. 5,399,346, 5,580,859, 5,589,466, incorporated by reference hereinin their entireties. In another embodiment, the invention provides agene therapy vector.

The nucleic acid can be cloned into a number of types of vectors. Forexample, the nucleic acid can be cloned into a vector including, but notlimited to a plasmid, a phagemid, a phage derivative, an animal virus,and a cosmid. Vectors of particular interest include expression vectors,replication vectors, probe generation vectors, and sequencing vectors.

Further, the expression vector may be provided to a cell in the form ofa viral vector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al. (2001, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York), and inother virology and molecular biology manuals. Viruses, which are usefulas vectors include, but are not limited to, retroviruses, adenoviruses,adeno-associated viruses, herpes viruses, and lentiviruses. In general,a suitable vector contains an origin of replication functional in atleast one organism, a promoter sequence, convenient restrictionendonuclease sites, and one or more selectable markers, (e.g., WO01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. A selected gene can be inserted intoa vector and packaged in retroviral particles using techniques known inthe art. The recombinant virus can then be isolated and delivered tocells of the subject either in vivo or ex vivo. A number of retroviralsystems are known in the art. In some embodiments, retrovirus vectorsare used. A number of retrovirus vectors are known in the art. In someembodiments, lentivirus vectors are used.

Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either cooperatively orindependently to activate transcription.

One example of a suitable promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Another example of a suitable promoter is Elongation Factor-1a (EF-1a).However, other constitutive promoter sequences may also be used,including, but not limited to the simian virus 40 (SV40) early promoter,mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV)long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemiavirus promoter, an Epstein-Barr virus immediate early promoter, a Roussarcoma virus promoter, as well as human gene promoters such as, but notlimited to, the actin promoter, the myosin promoter, the hemoglobinpromoter, and the creatine kinase promoter. Further, the inventionshould not be limited to the use of constitutive promoters. Induciblepromoters are also contemplated as part of the invention. The use of aninducible promoter provides a molecular switch capable of turning onexpression of the polynucleotide sequence which it is operatively linkedwhen such expression is desired, or turning off the expression whenexpression is not desired. Examples of inducible promoters include, butare not limited to a metallothionine promoter, a glucocorticoidpromoter, a progesterone promoter, and a tetracycline promoter. In someembodiments, the promoter is a EF-1a promoter.

In order to assess the expression of a CAR polypeptide or portionsthereof, the expression vector to be introduced into a cell can alsocontain either a selectable marker gene or a reporter gene or both tofacilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected through viralvectors. In other aspects, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection procedure. Bothselectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Usefulselectable markers include, for example, antibiotic-resistance genes,such as neo and the like, and fluorescent genes such as GFP, YFP, RFPand the like. In some embodiments, reporter genes or selectable markergenes are excluded from a CAR polypeptide used in a therapy as describedherein.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity, antibiotic resistance or fluorescence. Expression ofthe reporter gene is assayed at a suitable time after the DNA has beenintroduced into the recipient cells. Suitable reporter genes may includegenes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or the green fluorescentprotein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).Suitable expression systems are well known and may be prepared usingknown techniques or obtained commercially. In general, the constructwith the minimal 5′ flanking region showing the highest level ofexpression of reporter gene is identified as the promoter. Such promoterregions may be linked to a reporter gene and used to evaluate agents forthe ability to modulate promoter-driven transcription.

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans. In some embodiments, the host cell is a T cell.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al. (2001,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York). A preferred method for the introduction of a polynucleotideinto a host cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle).

In the case where a non-viral delivery system is utilized, an exemplarydelivery vehicle is a liposome. The use of lipid formulations iscontemplated for the introduction of the nucleic acids into a host cell(in vitro, ex vivo or in vivo). In another aspect, the nucleic acid maybe associated with a lipid. The nucleic acid associated with a lipid maybe encapsulated in the aqueous interior of a liposome, interspersedwithin the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome,dispersed in a solution containing a lipid, mixed with a lipid, combinedwith a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid,lipid/DNA or lipid/expression vector associated compositions are notlimited to any particular structure in solution. For example, they maybe present in a bilayer structure, as micelles, or with a “collapsed”structure. They may also simply be interspersed in a solution, possiblyforming aggregates that are not uniform in size or shape. Lipids arefatty substances which may be naturally occurring or synthetic lipids.For example, lipids include the fatty droplets that naturally occur inthe cytoplasm as well as the class of compounds which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids,alcohols, amines, amino alcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Chloroform is used as the only solventsince it is more readily evaporated than methanol. “Liposome” is ageneric term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,1991 Glycobiology 5: 505-10). However, compositions that have differentstructures in solution than the normal vesicular structure are alsoencompassed. For example, the lipids may assume a micellar structure ormerely exist as nonuniform aggregates of lipid molecules. Alsocontemplated are lipofectamine-nucleic acid complexes.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the inhibitor of the presentinvention, in order to confirm the presence of the recombinant DNAsequence in the host cell, a variety of assays may be performed. Suchassays include, for example, “molecular biological” assays well known tothose of skill in the art, such as Southern and Northern blotting,RT-PCR and PCR; “biochemical” assays, such as detecting the presence orabsence of a particular peptide, e.g., by immunological means (ELISAsand Western blots) or by assays described herein to identify agentsfalling within the scope of the invention.

RNA Transfection

In some embodiments, the genetically modified T cells of the inventionare modified through the introduction of RNA (e.g., an mRNA comprises asequence encoding a CAR as described herein). In some embodiments, an invitro transcribed RNA CAR can be introduced to a cell as a form oftransient transfection. The RNA is produced by in vitro transcriptionusing a polymerase chain reaction (PCR)-generated template. DNA ofinterest from any source can be directly converted by PCR into atemplate for in vitro mRNA synthesis using appropriate primers and RNApolymerase. The source of the DNA can be, for example, genomic DNA,plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any otherappropriate source of DNA. The desired template for in vitrotranscription is the CAR of the present invention. For example, in someembodiments, the template for the RNA CAR comprises an extracellulardomain comprising an anti-CD19 scFv; a transmembrane domain (such as thetransmembrane domain of CD28); and a cytoplasmic domain comprises thesignaling domain of CD3-zeta and the cytoplasmic domain of IL-15Rα. Insome embodiments, the template for the RNA CAR comprises anextracellular domain comprising an anti-CD30 scFv; a transmembranedomain (such as the transmembrane domain of CD28); and a cytoplasmicdomain comprises a CD27 intracellular domain, a 4-1BB intracellulardomain, and the signaling domain of CD3-zeta. In some embodiments, thetemplate for the RNA CAR comprises an extracellular domain comprising ananti-GD2 scFv; a transmembrane domain (such as the transmembrane domainof CD28); and a cytoplasmic domain comprises a CD27 intracellulardomain, a 4-1BB intracellular domain, and the signaling domain ofCD3-zeta.

RNA can be introduced into target cells using any of a number ofdifferent methods, for instance, commercially available methods whichinclude, but are not limited to, electroporation (Amaxa Nucleofector-II(Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (HarvardInstruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver,Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposomemediated transfection using lipofection, polymer encapsulation, peptidemediated transfection, or biolistic particle delivery systems such as“gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther.,12(8):861-70 (2001).

Genetically Modified Immune Cells

In some embodiments, the CAR sequence(s) (e.g., nucleic acid sequencesencoding a CAR as described herein) are delivered into cells (e.g., Tcells or NK cells) using a retroviral or lentiviral vector. In someembodiments, the arrangement of the elements of the CAR encoded by theCAR sequence(s) is selected from one of the following exemplary,non-limiting arrangements:

scFv-CD28-IL-15Rα-CD3zscFv-CD28-CD27-IL-15Rα-CD3zscFv-CD28-(4-1BB)-CD27-IL-15Rα-CD3zscFv-CD8-CD27-IL-15Rα-CD3zscFv-CD8-(4-1BB)-CD27-IL-15Rα-CD3zscFv-CD28-(4-1BB)-CD27-CD3z-iCasp9-FKBPscFv-CD28-CD27-(4-1BB)-CD3z-iCasp9-FKBPscFv-CD8-(4-1BB)-CD27-CD3z-iCasp9-FKBPscFv-CD8-CD27-(4-1BB)-CD3z-iCasp9-FKBP

GD2scFv-CD28-(4-1BB)-CD27-CD3z GD2scFv-CD8-CD28-CD3zGD2scFv-CD28-(4-1BB)-CD27-CD3z-Casp9-FKBPGD2scFv-CD8-CD28-CD3z-Casp9-FKBP

In some embodiments, the above exemplary, non-limiting arrangements arefrom left to right, N-terminus to C-terminus of the CAR. The CAR maycomprise or further comprise any other combination of elements asdescribed herein.

CAR-expressing retroviral and lentiviral vectors can be delivered intodifferent types of eukaryotic cells as well as into tissues and wholeorganisms using transduced cells as carriers or cell-free local orsystemic delivery of encapsulated, bound or naked vectors. The methodused can be for any purpose where stable expression is required orsufficient.

In another embodiment, the desired CAR can be expressed in the cells(e.g., T cells or NK cells) by way of transposons.

The disclosed methods can be applied to the modulation of immune cell(e.g., T cell or NK cell) activity in basic research and therapy, in thefields of cancer, stem cells, acute and chronic infections, andautoimmune diseases, including the assessment of the ability of thegenetically modified T cell or NK cell to kill a target cell, e.g., atarget cancer cell.

The methods also provide the ability to control the level of expressionover a wide range by changing, for example, the promoter or the amountof input vector, making it possible to individually regulate theexpression level. For example, varying of different intracellulareffector/costimulator domains on multiple chimeric receptors in the samecell allows determination of the structure of the receptor combinationswhich assess the highest level of cytotoxicity against multi-antigenictargets, and at the same time lowest cytotoxicity toward normal cells.

Sources of Immune Cells

Prior to expansion and genetic modification of the immune cells (e.g., Tcells) of the invention, a source of immune cells (e.g., T cells) isobtained from a subject. Immune cells (e.g., T cells) can be obtainedfrom a number of sources, including peripheral blood mononuclear cells,bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from asite of infection, ascites, pleural effusion, spleen tissue, and tumors.The immune cells (e.g., T cells) may also be generated from inducedpluripotent stem cells or hematopoietic stem cells or progenitor cells.In some embodiments of the present invention, any number of immune celllines, including but not limited to T cell and NK cell lines, availablein the art, may be used. In some embodiments of the present invention,immune cells (e.g., T cells) can be obtained from a unit of bloodcollected from a subject using any number of techniques known to theskilled artisan, such as Ficoll™ separation. In some embodiments, cellsfrom the circulating blood of an individual are obtained by apheresis.The apheresis product typically contains lymphocytes, including T cells,monocytes, granulocytes, B cells, NK cells, other nucleated white bloodcells, red blood cells, and platelets. In some embodiments, the cellscollected by apheresis may be washed to remove the plasma fraction andto place the cells in an appropriate buffer or media for subsequentprocessing steps. In some embodiments of the invention, the cells arewashed with phosphate buffered saline (PBS). In an alternativeembodiment, the wash solution lacks calcium and may lack magnesium ormay lack many if not all divalent cations. Again, surprisingly, initialactivation steps in the absence of calcium lead to magnified activation.As those of ordinary skill in the art would readily appreciate a washingstep may be accomplished by methods known to those in the art, such asby using a semi-automated “flow-through” centrifuge (for example, theCobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics CellSaver 5) according to the manufacturer's instructions. After washing,the cells may be resuspended in a variety of biocompatible buffers, suchas, for example, Ca²⁺-free, Mg²⁺-free PBS, PlasmaLyte A, or other salinesolution with or without buffer. Alternatively, the undesirablecomponents of the apheresis sample may be removed and the cells directlyresuspended in culture media.

In another embodiment, immune cells (e.g., T cells) are isolated fromperipheral blood lymphocytes by lysing the red blood cells and depletingthe monocytes, for example, by centrifugation through a PERCOLL™gradient or by counterflow centrifugal elutriation. A specificsubpopulation of T cells, such as CD3⁺, CD28⁺, CD4⁺, CD8⁺, CD45RA⁺, andCD45RO⁺ T cells, can be further isolated by positive or negativeselection techniques. For example, in some embodiments, T cells areisolated by incubation with anti-CD3/anti-CD28 (i.e., 3×28)-conjugatedbeads, such as DYNABEADS® M-450 CD3/CD28 T, for a time period sufficientfor positive selection of the desired T cells. In some embodiments, thetime period is about 30 minutes. In a further embodiment, the timeperiod ranges from 30 minutes to 36 hours or longer and all integervalues there between. In a further embodiment, the time period is atleast 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment,the time period is 10 to 24 hours. In one preferred embodiment, theincubation time period is 24 hours. For isolation of T cells frompatients with leukemia, use of longer incubation times, such as 24hours, can increase cell yield. Longer incubation times may be used toisolate T cells in any situation where there are few T cells as comparedto other cell types, such in isolating tumor infiltrating lymphocytes(TIL) from tumor tissue or from immune-compromised individuals. Further,use of longer incubation times can increase the efficiency of capture ofCD8+ T cells. Thus, by simply shortening or lengthening the time T cellsare allowed to bind to the CD3/CD28 beads and/or by increasing ordecreasing the ratio of beads to T cells (as described further herein),subpopulations of T cells can be preferentially selected for or againstat culture initiation or at other time points during the process.Additionally, by increasing or decreasing the ratio of anti-CD3 and/oranti-CD28 antibodies on the beads or other surface, subpopulations of Tcells can be preferentially selected for or against at cultureinitiation or at other desired time points. The skilled artisan wouldrecognize that multiple rounds of selection can also be used in thecontext of this invention. In certain embodiments, it may be desirableto perform the selection procedure and use the “unselected” cells in theactivation and expansion process. “Unselected” cells can also besubjected to further rounds of selection.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. One method is cellsorting and/or selection via negative magnetic immunoadherence or flowcytometry that uses a cocktail of monoclonal antibodies directed to cellsurface markers present on the cells negatively selected. For example,to enrich for CD4⁺ cells by negative selection, a monoclonal antibodycocktail typically includes antibodies to CD14, CD20, CD1 1b, CD16,HLA-DR, and CD8. In certain embodiments, it may be desirable to enrichfor or positively select for regulatory T cells which typically expressCD4⁺, CD25⁺, CD62L^(hi), GITR⁺, and FoxP3⁺.

Alternatively, in certain embodiments, T regulatory cells are depletedby anti-C25 conjugated beads or other similar method of selection.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain embodiments, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in some embodiments, aconcentration of 2 billion cells/ml is used. In some embodiments, aconcentration of 1 billion cells/ml is used. In a further embodiment,greater than 100 million cells/ml is used. In a further embodiment, aconcentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 millioncells/ml is used. In yet another embodiment, a concentration of cellsfrom 75, 80, 85, 90, 95, or 100 million cells/ml is used. In furtherembodiments, concentrations of 125 or 150 million cells/ml can be used.Using high concentrations can result in increased cell yield, cellactivation, and cell expansion. Further, use of high cell concentrationsallows more efficient capture of cells that may weakly express targetantigens of interest, such as CD28-negative T cells, or from sampleswhere there are many tumor cells present (e.g., leukemic blood, tumortissue, etc.). Such populations of cells may have therapeutic value andwould be desirable to obtain. For example, using high concentration ofcells allows more efficient selection of CD8⁺ T cells that normally haveweaker CD28 expression.

In a related embodiment, it may be desirable to use lower concentrationsof cells. By significantly diluting the mixture of T cells and surface(e.g., particles such as beads), interactions between the particles andcells is minimized. This selects for cells that express high amounts ofdesired antigens to be bound to the particles. For example, CD4⁺ T cellsexpress higher levels of CD28 and are more efficiently captured thanCD8⁺ T cells in dilute concentrations. In some embodiments, theconcentration of cells used is 5×10⁶/ml. In other embodiments, theconcentration used can be from about 1×10⁵/ml to 1×10⁶/ml, and anyinteger value in between.

In other embodiments, the cells may be incubated on a rotator forvarying lengths of time at varying speeds at either 2-10° C. or at roomtemperature.

T cells for stimulation can also be frozen after a washing step. Wishingnot to be bound by theory, the freeze and subsequent thaw step providesa more uniform product by removing granulocytes and to some extentmonocytes in the cell population. After the washing step that removesplasma and platelets, the cells may be suspended in a freezing solution.While many freezing solutions and parameters are known in the art andwill be useful in this context, one method involves using PBS containing20% DMSO and 8% human serum albumin, or culture media containing 10%Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitablecell freezing media containing for example, Hespan and PlasmaLyte A, thecells then are frozen to −80° C. at a rate of 1° per minute and storedin the vapor phase of a liquid nitrogen storage tank. Other methods ofcontrolled freezing may be used as well as uncontrolled freezingimmediately at −20° C. or in liquid nitrogen. In certain embodiments,cryopreserved cells are thawed and washed as described herein andallowed to rest for one hour at room temperature prior to activationusing the methods of the present invention.

Also contemplated in the context of the invention is the collection ofblood samples or apheresis product from a subject at a time period priorto when the expanded cells as described herein might be needed. As such,the source of the cells to be expanded can be collected at any timepoint necessary, and desired cells, such as T cells, isolated and frozenfor later use in T cell therapy for any number of diseases or conditionsthat would benefit from T cell therapy, such as those described herein.In some embodiments a blood sample or an apheresis is taken from agenerally healthy subject. In certain embodiments, a blood sample or anapheresis is taken from a generally healthy subject who is at risk ofdeveloping a disease, but who has not yet developed a disease, and thecells of interest are isolated and frozen for later use. In certainembodiments, the T cells may be expanded, frozen, and used at a latertime. In certain embodiments, samples are collected from a patientshortly after diagnosis of a particular disease as described herein butprior to any treatments. In a further embodiment, the cells are isolatedfrom a blood sample or an apheresis from a subject prior to any numberof relevant treatment modalities, including but not limited to treatmentwith agents such as natalizumab, efalizumab, antiviral agents,chemotherapy, radiation, immunosuppressive agents, such as cyclosporin,azathioprine, methotrexate, mycophenolate, and FK506, antibodies, orother immunoablative agents such as CAMPATH, anti-CD3 antibodies,Cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid,steroids, FR901228, and irradiation. These drugs inhibit either thecalcium dependent phosphatase calcineurin (cyclosporine and FK506) orinhibit the p70S6 kinase that is important for growth factor inducedsignaling (rapamycin) (Liu et al., Cell 66:807-815, 1991; Henderson etal., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun.5:763-773, 1993). In a further embodiment, the cells are isolated for apatient and frozen for later use in conjunction with (e.g., before,simultaneously or following) bone marrow or stem cell transplantation, Tcell ablative therapy using either chemotherapy agents such as,fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, orantibodies such as OKT3 or CAMPATH. In another embodiment, the cells areisolated prior to and can be frozen for later use for treatmentfollowing B-cell ablative therapy such as agents that react with CD20,e.g., Rituxan.

In a further embodiment of the present invention, T cells are obtainedfrom a patient directly following treatment. In this regard, it has beenobserved that following certain cancer treatments, in particulartreatments with drugs that damage the immune system, shortly aftertreatment during the period when patients would normally be recoveringfrom the treatment, the quality of T cells obtained may be optimal orimproved for their ability to expand ex vivo. Likewise, following exvivo manipulation using the methods described herein, these cells may bein a preferred state for enhanced engraftment and in vivo expansion.Thus, it is contemplated within the context of the present invention tocollect blood cells, including T cells, dendritic cells, or other cellsof the hematopoietic lineage, during this recovery phase. Further, incertain embodiments, mobilization (for example, mobilization withGM-CSF) and conditioning regimens can be used to create a condition in asubject wherein repopulation, recirculation, regeneration, and/orexpansion of particular cell types is favored, especially during adefined window of time following therapy. Illustrative cell typesinclude T cells, B cells, dendritic cells, and other cells of the immunesystem.

Activation and Expansion of T Cells

Whether prior to or after genetic modification of the T cells to expressa desirable CAR, the T cells can be activated and expanded generallyusing methods as described, for example, in U.S. Pat. Nos. 6,352,694 and6,534,055.

Generally, the T cells of the invention are expanded by contact with asurface having attached thereto an agent that stimulates a CD3/TCRcomplex associated signal and a ligand that stimulates a co-stimulatorymolecule on the surface of the T cells. In particular, T cellpopulations may be stimulated as described herein, such as by contactwith an anti-CD3 antibody, or antigen-binding fragment thereof, or ananti-CD2 antibody immobilized on a surface, or by contact with a proteinkinase C activator (e.g., bryostatin) in conjunction with a calciumionophore. For co-stimulation of an accessory molecule on the surface ofthe T cells, a ligand that binds the accessory molecule is used. Forexample, a population of T cells can be contacted with an anti-CD3antibody and an anti-CD28 antibody, under conditions appropriate forstimulating proliferation of the T cells. To stimulate proliferation ofeither CD4⁺ T cells or CD8⁺ T cells, an anti-CD3 antibody and ananti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3,XR-CD28 (Diaclone, Besancon, France) can be used as can other methodscommonly known in the art (Berg et al., Transplant Proc.30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9): 13191328,1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999).

In certain embodiments, the primary stimulatory signal and theco-stimulatory signal for the T cell may be provided by differentprotocols. For example, the agents providing each signal may be insolution or coupled to a surface. When coupled to a surface, the agentsmay be coupled to the same surface (i.e., in “cis” formation) or toseparate surfaces (i.e., in “trans” formation). Alternatively, one agentmay be coupled to a surface and the other agent in solution. In someembodiments, the agent providing the co-stimulatory signal is bound to acell surface and the agent providing the primary activation signal is insolution or coupled to a surface. In certain embodiments, both agentscan be in solution. In another embodiment, the agents may be in solubleform, and then cross-linked to a surface, such as a cell expressing Fcreceptors or an antibody or other binding agent which will bind to theagents. In this regard, see for example, U.S. Patent ApplicationPublication Nos. 20040101519 and 20060034810 for artificial antigenpresenting cells (aAPCs) that are contemplated for use in activating andexpanding T cells in the present invention.

In some embodiments, the two agents are immobilized on beads, either onthe same bead, i.e., “cis,” or to separate beads, i.e., “trans.” By wayof example, the agent providing the primary activation signal is ananti-CD3 antibody or an antigen-binding fragment thereof and the agentproviding the co-stimulatory signal is an anti-CD28 antibody orantigen-binding fragment thereof; and both agents are co-immobilized tothe same bead in equivalent molecular amounts. In some embodiments, a1:1 ratio of each antibody bound to the beads for CD4⁺ T cell expansionand T cell growth is used. In certain aspects of the present invention,a ratio of anti CD3:CD28 antibodies bound to the beads is used such thatan increase in T cell expansion is observed as compared to the expansionobserved using a ratio of 1:1. In one particular embodiment an increaseof from about 1 to about 3 fold is observed as compared to the expansionobserved using a ratio of 1:1. In some embodiments, the ratio ofCD3:CD28 antibody bound to the beads ranges from 100:1 to 1:100 and allinteger values there between. In one aspect of the present invention,more anti-CD28 antibody is bound to the particles than anti-CD3antibody, i.e., the ratio of CD3:CD28 is less than one. In certainembodiments of the invention, the ratio of anti CD28 antibody to antiCD3 antibody bound to the beads is greater than 2:1. In one particularembodiment, a 1:100 CD3:CD28 ratio of antibody bound to beads is used.In another embodiment, a 1:75 CD3:CD28 ratio of antibody bound to beadsis used. In a further embodiment, a 1:50 CD3:CD28 ratio of antibodybound to beads is used. In another embodiment, a 1:30 CD3:CD28 ratio ofantibody bound to beads is used. In one preferred embodiment, a 1:10CD3:CD28 ratio of antibody bound to beads is used. In anotherembodiment, a 1:3 CD3:CD28 ratio of antibody bound to the beads is used.In yet another embodiment, a 3:1 CD3:CD28 ratio of antibody bound to thebeads is used.

Ratios of particles to cells from 1:500 to 500:1 and any integer valuesin between may be used to stimulate T cells or other target cells. Asthose of ordinary skill in the art can readily appreciate, the ratio ofparticles to cells may depend on particle size relative to the targetcell. For example, small sized beads could only bind a few cells, whilelarger beads could bind many. In certain embodiments the ratio of cellsto particles ranges from 1:100 to 100:1 and any integer valuesin-between and in further embodiments the ratio comprises 1:9 to 9:1 andany integer values in between, can also be used to stimulate T cells.The ratio of anti-CD3- and anti-CD28-coupled particles to T cells thatresult in T cell stimulation can vary as noted above, however certainpreferred values include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8,1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1,9:1, 10:1, and 15:1 with one preferred ratio being at least 1:1particles per T cell. In some embodiments, a ratio of particles to cellsof 1:1 or less is used. In one particular embodiment, a preferredparticle:cell ratio is 1:5. In further embodiments, the ratio ofparticles to cells can be varied depending on the day of stimulation.For example, in some embodiments, the ratio of particles to cells isfrom 1:1 to 10:1 on the first day and additional particles are added tothe cells every day or every other day thereafter for up to 10 days, atfinal ratios of from 1:1 to 1:10 (based on cell counts on the day ofaddition). In one particular embodiment, the ratio of particles to cellsis 1:1 on the first day of stimulation and adjusted to 1:5 on the thirdand fifth days of stimulation. In another embodiment, particles areadded on a daily or every other day basis to a final ratio of 1:1 on thefirst day, and 1:5 on the third and fifth days of stimulation. Inanother embodiment, the ratio of particles to cells is 2:1 on the firstday of stimulation and adjusted to 1:10 on the third and fifth days ofstimulation. In another embodiment, particles are added on a daily orevery other day basis to a final ratio of 1:1 on the first day, and 1:10on the third and fifth days of stimulation. One of skill in the art willappreciate that a variety of other ratios may be suitable for use in thepresent invention. In particular, ratios will vary depending on particlesize and on cell size and type.

In further embodiments of the present invention, the cells, such as Tcells, are combined with agent-coated beads, the beads and the cells aresubsequently separated, and then the cells are cultured. In analternative embodiment, prior to culture, the agent-coated beads andcells are not separated but are cultured together. In a furtherembodiment, the beads and cells are first concentrated by application ofa force, such as a magnetic force, resulting in increased ligation ofcell surface markers, thereby inducing cell stimulation.

By way of example, cell surface proteins may be ligated by allowingparamagnetic beads to which anti-CD3 and anti-CD28 are attached (3×28beads) to contact the T cells. In some embodiments the cells (forexample, 10⁴ to 10⁹ T cells) and beads (for example, DYNABEADS® M-450CD3/CD28 T paramagnetic beads at a ratio of 1:1) are combined in abuffer, preferably PBS (without divalent cations such as, calcium andmagnesium). Again, those of ordinary skill in the art can readilyappreciate any cell concentration may be used. For example, the targetcell may be very rare in the sample and comprise only 0.01% of thesample or the entire sample (i.e., 100%) may comprise the target cell ofinterest. Accordingly, any cell number is within the context of thepresent invention. In certain embodiments, it may be desirable tosignificantly decrease the volume in which particles and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and particles. For example, in some embodiments, aconcentration of about 2 billion cells/ml is used. In anotherembodiment, greater than 100 million cells/ml is used. In a furtherembodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45,or 50 million cells/ml is used. In yet another embodiment, aconcentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mlis used. In further embodiments, concentrations of 125 or 150 millioncells/ml can be used. Using high concentrations can result in increasedcell yield, cell activation, and cell expansion. Further, use of highcell concentrations allows more efficient capture of cells that mayweakly express target antigens of interest, such as CD28-negative Tcells. Such populations of cells may have therapeutic value and would bedesirable to obtain in certain embodiments. For example, using highconcentration of cells allows more efficient selection of CD8+ T cellsthat normally have weaker CD28 expression.

In some embodiments of the present invention, the mixture may becultured for several hours (about 3 hours) to about 14 days or anyhourly integer value in between. In another embodiment, the mixture maybe cultured for 21 days. In some embodiments of the invention the beadsand the T cells are cultured together for about eight days. In anotherembodiment, the beads and T cells are cultured together for 2-3 days.Several cycles of stimulation may also be desired such that culture timeof T cells can be 60 days or more. Conditions appropriate for T cellculture include an appropriate media (e.g., Minimal Essential Media orRPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factorsnecessary for proliferation and viability, including serum (e.g., fetalbovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4,IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFp, and TNF-α or any otheradditives for the growth of cells known to the skilled artisan. Otheradditives for the growth of cells include, but are not limited to,surfactant, plasmanate, and reducing agents such as N-acetyl-cysteineand 2-mercaptoethanol. Media can include RPMI 1640, AIM-V, DMEM, MEM,a-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added aminoacids, sodium pyruvate, and vitamins, either serum-free or supplementedwith an appropriate amount of serum (or plasma) or a defined set ofhormones, and/or an amount of cytokine(s) sufficient for the growth andexpansion of T cells. Antibiotics, e.g., penicillin and streptomycin,are included only in experimental cultures, not in cultures of cellsthat are to be infused into a subject. The target cells are maintainedunder conditions necessary to support growth, for example, anappropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5%C0₂).

T cells that have been exposed to varied stimulation times may exhibitdifferent characteristics. For example, typical blood or apheresedperipheral blood mononuclear cell products have a helper T cellpopulation (¾, CD4⁺) that is greater than the cytotoxic or suppressor Tcell population (T_(c), CD8⁺). Ex vivo expansion of T cells bystimulating CD3 and CD28 receptors produces a population of T cells thatprior to about days 8-9 consists predominately of ¾ cells, while afterabout days 8-9, the population of T cells comprises an increasinglygreater population of Tc cells. Accordingly, depending on the purpose oftreatment, infusing a subject with a T cell population comprisingpredominately of T_(H) cells may be advantageous. Similarly, if anantigen-specific subset of Tc cells has been isolated it may bebeneficial to expand this subset to a greater degree.

Further, in addition to CD4 and CD 8 markers, other phenotypic markersvary significantly, but in large part, reproducibly during the course ofthe cell expansion process. Thus, such reproducibility enables theability to tailor an activated T cell product for specific purposes.

Therapeutic Application

In some embodiments, the present invention provides a cell (e.g., Tcell) modified to express a CAR comprises an antigen binding domain, atransmembrane domain (such as CD28 transmembrane domain), and acytoplasmic domain comprising an IL-15Rα cytoplasmic domain, optionallycombined with CD3-zeta and/or any other cytoplasmic domains describedherein. In some embodiments, a cell is modified to express a CARcomprising an antigen binding domain, a transmembrane domain (such asCD28 transmembrane domain), and a cytoplasmic domain having a CD27intracellular domain and a 4-1BB intracellular domain, optionallycombined with CD3-zeta and/or any other cytoplasmic domains (e.g., aniCasp9-FKBP domain) described herein. In some embodiments, a cell ismodified to express a CAR comprising an antigen binding domain (e.g., ascFV specific for GD2), a transmembrane domain (such as CD28transmembrane domain), and a cytoplasmic domain. Therefore, in someinstances, the transduced T cell can elicit a CAR-mediated T-cellresponse. In some embodiments, the invention provides the use of a CARto redirect the specificity of a primary T cell to an antigen, such as atumor antigen. Thus, in some embodiments, the present invention alsoprovides a method for stimulating a T cell-mediated immune response to atarget cell population or tissue in a mammal comprising the step ofadministering to the mammal a T cell that expresses a CAR, wherein theCAR comprises an antigen binding domain (e.g., CD19 scFV), atransmembrane domain (such as CD28 transmembrane domain), and acytoplasmic domain comprising an IL-15Rα cytoplasmic domain, optionallycombined with CD3-zeta and/or any other cytoplasmic domains describedherein.

In some embodiments, a method for stimulating a T cell-mediated immuneresponse to a target cell population or tissue in a mammal comprisingthe step of administering to the mammal a T cell that expresses a CAR,wherein the CAR comprises an antigen binding domain (e.g., CD30 scFV), atransmembrane domain (such as CD28 transmembrane domain), and acytoplasmic domain having a CD27 intracellular domain and a 4-1BBintracellular domain, optionally combined with CD3-zeta and/or any othercytoplasmic domains (e.g., iCasp9 domain and FKBP domain) describedherein.

In some embodiments, the disclosure provides a method for stimulating aT cell-mediated immune response to a target cell population or tissue ina mammal comprising the step of administering to the mammal a T cellthat expresses a CAR, wherein the CAR comprises an antigen bindingdomain (e.g., GD2 scFV), a transmembrane domain (such as CD28transmembrane domain), and a cytoplasmic domain (e.g., comprising aCD28, 4-1BB, and/or CD27 intracellular domain combined with CD3-zetaand/or any other cytoplasmic domains described herein.

In some embodiments, the present invention includes a type of cellulartherapy where T cells are genetically modified to express a CAR and theCAR T cell is infused to a recipient in need thereof. The infused cellis able to kill cells expressing the antigen, e.g., tumor cells, in therecipient. Unlike antibody therapies, CAR T cells are able to replicatein vivo resulting in long-term persistence that can lead to sustainedtumor control.

In some embodiments, the CAR T cells of the invention can undergo robustin vivo T cell expansion and can persist for an extended amount of time.

While the data disclosed herein specifically disclose lentiviral vectorcomprising (1) anti-CD19 scFv, a CD28 transmembrane domain, a IL-15Rαcytoplasmic domain and a CD3-zeta signaling domain, (2) anti-CD30 scFv,a CD28 transmembrane domain, a cytoplasmic domain having a CD27intracellular domain and a 4-1BB intracellular domain, and a CD3-zetasignaling domain, and (3) anti-GD2 scFv, a CD28 transmembrane domain, acytoplasmic domain having a CD27 intracellular domain and a 4-1BBintracellular domain, and a CD3-zeta signaling domain, the inventionshould be construed to include any number of variations for each of thecomponents of the construct as described elsewhere herein. That is, theinvention includes the use of any antigen binding domain in the CAR togenerate a CAR-mediated T-cell response specific to the antigen bindingdomain. For example, the antigen binding domain in the CAR of theinvention can target a tumor antigen for the purposes of treat cancer.

In some embodiments, the antigen bind domain portion of the CAR of theinvention is designed to treat a particular cancer. For example, the CARmay be designed to target CD19 for treating B cell malignancies, or CD30for treating Hodgkin's lymphoma or certain T cell lymphoma, or GD2 fortreating small cell neuroendocrine cancer or small cell lung cancer, andneuronal cancer.

The CAR-modified T cells of the invention may also serve as a type ofvaccine for ex vivo immunization and/or in vivo therapy in a mammal.Preferably, the mammal is a human.

With respect to ex vivo immunization, at least one of the followingoccurs in vitro prior to administering the cell into a mammal: i)expansion of the cells, ii) introducing a nucleic acid encoding a CAR tothe cells, and/or iii) cryopreservation of the cells.

Ex vivo procedures are well known in the art and are discussed morefully below. Briefly, cells are isolated from a mammal (preferably ahuman) and genetically modified (e.g., transduced or transfected invitro) with a vector expressing a CAR disclosed herein. The CAR-modifiedcell can be administered to a mammalian recipient to provide atherapeutic benefit. The mammalian recipient may be a human and theCAR-modified cell can be autologous with respect to the recipient.Alternatively, the cells can be allogeneic, syngeneic or xenogeneic withrespect to the recipient.

The procedure for ex vivo expansion of hematopoietic stem and progenitorcells is described in U.S. Pat. No. 5,199,942, incorporated herein byreference, can be applied to the cells of the present invention. Othersuitable methods are known in the art, therefore the present inventionis not limited to any particular method of ex vivo expansion of thecells. Briefly, ex vivo culture and expansion of T cells comprises: (1)collecting CD34+ hematopoietic stem and progenitor cells from a mammalfrom peripheral blood harvest or bone marrow explants; and (2) expandingsuch cells ex vivo. In addition to the cellular growth factors describedin U.S. Pat. No. 5,199,942, other factors such as flt3-L, IL-1, IL-3 andc-kit ligand, can be used for culturing and expansion of the cells. Inaddition to using a cell-based vaccine in terms of ex vivo immunization,the present invention also provides compositions and methods for in vivoimmunization to elicit an immune response directed against an antigen ina patient.

Generally, the cells activated and expanded as described herein may beutilized in the treatment and prevention of diseases that arise inindividuals who are immunocompromised, such as individuals havingcancer.

The CAR-modified immune cells (e.g., CAR T cells) of the presentinvention may be administered either alone, or as a composition (e.g., apharmaceutical composition) in combination with diluents and/or withother components such as IL-2 or other cytokines or cell populations.Briefly, pharmaceutical compositions of the present invention maycomprise a target cell population as described herein, in combinationwith one or more pharmaceutically or physiologically acceptablecarriers, diluents or excipients. Such compositions may comprise bufferssuch as neutral buffered saline, phosphate buffered saline and the like;carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol;proteins; polypeptides or amino acids such as glycine; antioxidants;chelating agents such as EDTA or glutathione; adjuvants {e.g., aluminumhydroxide); and preservatives.

Compositions of the present invention are preferably formulated forintravenous administration.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials.

When “therapeutic amount” is indicated, the precise amount of thecompositions of the present invention to be administered can bedetermined by a physician with consideration of individual differencesin age, weight, tumor size, extent of infection or metastasis, andcondition of the patient (subject). It can generally be stated that apharmaceutical composition comprising the CAR-modified immune cells(e.g., CAR T cells) described herein may be administered at a dosage of10⁴ to 10⁹ cells/kg body weight, preferably 10⁵ to 10⁶ cells/kg bodyweight, including all integer values within those ranges. T cellcompositions may also be administered multiple times at these dosages.The cells can be administered by using infusion techniques that arecommonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng.J. of Med. 319: 1676, 1988). The optimal dosage and treatment regime fora particular patient can readily be determined by one skilled in the artof medicine by monitoring the patient for signs of disease and adjustingthe treatment accordingly.

In certain embodiments, it may be desired to administer activated immune(e.g., T cells) to a subject and then subsequently redraw blood (or havean apheresis performed), activate T cells therefrom according to thepresent invention, and reinfuse the patient with these activated andexpanded T cells. This process can be carried out multiple times everyfew weeks. In certain embodiments, T cells can be activated from blooddraws of from 10cc to 400cc. In certain embodiments, T cells areactivated from blood draws of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc,90cc, or 100cc. Not to be bound by theory, using this multiple blooddraw/multiple reinfusion protocol may serve to select out certainpopulations of T cells.

The administration of the subject compositions may be carried out in anyconvenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein may be administered to a patientsubcutaneously, intradermally, intratumorally, intranodally,intramedullary, intramuscularly, by intravenous (i.v.) injection, orintraperitoneally. In some embodiments, the T cell compositions of thepresent invention are administered to a patient by intradermal orsubcutaneous injection. In another embodiment, the T cell compositionsof the present invention are preferably administered by i.v. injection.The compositions of T cells may be injected directly into a tumor, lymphnode, or site of infection.

In certain embodiments of the present invention, cells activated andexpanded using the methods described herein, or other methods known inthe art where T cells are expanded to therapeutic levels, areadministered to a patient in conjunction with (e.g., before,simultaneously or following) any number of relevant treatmentmodalities, including but not limited to treatment with agents such asantiviral therapy, cidofovir and interleukin-2, Cytarabine (also knownas ARA-C) or natalizumab treatment for MS patients or efalizumabtreatment for psoriasis patients or other treatments for PML patients.In further embodiments, the T cells of the invention may be used incombination with chemotherapy, radiation, immunosuppressive agents, suchas cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506,antibodies, or other immunoablative agents such as CAM PATH, anti-CD3antibodies or other antibody therapies, cytoxin, fludaribine,cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228,cytokines, and irradiation. These drugs inhibit either the calciumdependent phosphatase calcineurin (cyclosporine and FK506) or inhibitthe p70S6 kinase that is important for growth factor induced signaling(rapamycin). In a further embodiment, the cell compositions of thepresent invention are administered to a patient in conjunction with(e.g., before, simultaneously or following) bone marrow transplantation,T cell ablative therapy using either chemotherapy agents such as,fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, orantibodies such as OKT3 or CAMPATH. In another embodiment, the cellcompositions of the present invention are administered following B-cellablative therapy such as agents that react with CD20, e.g., Rituxan. Forexample, in some embodiments, subjects may undergo standard treatmentwith high dose chemotherapy followed by peripheral blood stem celltransplantation. In certain embodiments, following the transplant,subjects receive an infusion of the expanded immune cells of the presentinvention. In an additional embodiment, expanded cells are administeredbefore or following surgery.

In certain embodiments of the present invention, cells activated andexpanded using the methods described herein, or other methods known inthe art where T cells are expanded to therapeutic levels, or any othercompositions described herein, are administered to a patient inconjunction with (e.g., before, simultaneously or following) any numberof relevant treatment modalities, including checkpoint inhibitors, suchas PD-L1 inhibitors or PD1 inhibitors. In some embodiments, the PD-L1inhibitors or PD1 inhibitors are PD-L1-specific antibodies orPD1-specific antibodies. Exemplary checkpoint inhibitors include, e.g.,pembrolizumab (Merck), ipilimumab (Bristol-Myers Squibb), nivolumab(Bristol-Myers Squibb), MPDL3280A (Roche), MEDI4736 (AstraZeneca),MEDI0680 (AstraZeneca), BMS-936559/MDX-1105 (Bristol-Myers Squibb) andMSB0010718C (Merck). Other PD-L1 and PD1 inhibitors are known in the art(see, e.g., Dolan et al. PD-1 pathway inhibitors: changing the landscapeof cancer immunotherapy. Cancer Control. 2014 July; 21(3):231-7). Insome embodiments, compositions described herein are administered inconjunction with (e.g., before, simultaneously or following)chemotherapy and/or radiotherapy.

The dosage of the above treatments to be administered to a patient willvary with the precise nature of the condition being treated and therecipient of the treatment. The scaling of dosages for humanadministration can be performed according to art-accepted practices. Thedose for CAMPATH, for example, will generally be in the range 1 to about100 mg for an adult patient, usually administered daily for a periodbetween 1 and 30 days. The preferred daily dose is 1 to 10 mg per dayalthough in some instances larger doses of up to 40 mg per day may beused (described in U.S. Pat. No. 6,120,766). Strategies for CAR T celldosing and scheduling have been discussed (Ertl et al, 2011, Cancer Res,71:3175-81; Junghans, 2010, Journal of Translational Medicine, 8:55).

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present disclosure toits fullest extent. The following specific embodiments are, therefore,to be construed as merely illustrative, and not limitative of theremainder of the disclosure in any way whatsoever. All publicationscited herein are incorporated by reference for the purposes or subjectmatter referenced herein.

EXAMPLES Example 1: Interleukin 15Rα as Co-Stimulatory Domain in CD19CART Cells Enhances the Anti-Leukemia Efficacy

Chimeric Antigen Receptor T cells (CARTs) are engineered T cellsdisplaying specificity against tumor antigens, usually based on one ormore single chain Fv (scFv) antibody moieties. The initial CAR designconsisted of a receptor complex that combined an antigen binding singlechain antibody Fv domain (scFv) and a signal transduction domain of Tcells (usually CD3) {Eshhar Z, 1993, Specific activation and targetingof cytotoxic lymphocytes through chimeric single chains consisting ofantibody-binding domains and gamma or zeta subunits of theimmunoglobulins or T cell receptors}. These so called first generationCARs have only limited anti-tumor activities and in vivo survival{Kowolik CM1, 2006, CD28 costimulation provided through a CD19-specificchimeric antigen receptor enhances in vivo persistence and antitumorefficacy of adoptively transferred T cells.; Hwu P1, 1995, In vivoantitumor activity of T cells redirected with chimeric antibody/T-cellreceptor genes.}. In order to enhance killing efficacy and persistenceof CARTs, several second and third generation CARs with the addition ofone or two co-stimulatory signals such as CD28 and 4-1BB have beengenerated {Kowolik CM1, 2006, CD28 costimulation provided through aCD19-specific chimeric antigen receptor enhances in vivo persistence andantitumor efficacy of adoptively transferred T cells.; Savoldo B, 2011,CD28 costimulation improves expansion and persistence of chimericantigen receptor-modified T cells in lymphoma patients.; Song D G, 2011,In vivo persistence, tumor localization, and antitumor activity ofCAR-engineered T cells is enhanced by costimulatory signaling throughCD137 (4-1BB).; Milone M C, 2009, Chimeric receptors containing CD137signal transduction domains mediate enhanced survival of T cells andincreased antileukemic efficacy in vivo.}.

CD19 has proved to be an effective tumor antigen to target B cellmalignancies using CARTs. The first success with CD19 targeting CARTswas reported in chronic lymphocytic leukemia patients and adults withrefractory B cell leukemias {Porter D L, 2011, Chimeric antigenreceptor-modified T cells in chronic lymphoid leukemia.; Brentjens R J,2013, CD19-targeted T cells rapidly induce molecular remissions inadults with chemotherapy-refractory acute lymphoblastic leukemia.}. Thefield is moving extremely fast and recently found to be useful inpediatric acute B cell leukemia {Grupp S A, 2013, Chimeric AntigenReceptor-Modified T cells for Acute Lymphoid Leukemia.}. Preliminaryresults of CD19 targeted CART clinical trials indicate that this therapyis effective in various settings including post hematopoietic stem celltransplant relapsed leukemia using donor T cells engineered withCD19-CAR {Cruz C R, 2013, Infusion of donor-derived CD19-redirectedvirus-specific T cells for B-cell malignancies relapsed after allogeneicstem cell transplant: a phase 1 study.; Brentjens R J, 2011, Safety andpersistence of adoptively transferred autologous CD19-targeted T cellsin patients with relapsed or chemotherapy refractory B-cell leukemias.;Kochenderfer J N, 2013, Donor-derived CD19-targeted T cells causeregression of malignancy persisting after allogeneic hematopoietic stemcell transplantation.}.

Results of the CART clinical trials conducted so far indicate thatseveral factors may be influencing the performance of CARTs includingthose related to disease, patient-related factors and last but notleast, CART characteristics. The current focus is on generating CARTswith increased efficacy, longer survival and a good safety profile sothat they can be applied to a wider range of malignancies includingsolid tumors which harbor a hostile tumor microenvironment {Maus M V,2014, Antibody-modified T cells: CARs take the front seat forhematologic malignancies.; CJ., 2014, Chimeric antigen receptor modifiedT cell therapy for B cell malignancies.}. One of the ways this can beaccomplished is by potentiating CAR signaling and activation of thegenetically modified T cells.

Interleukin-15 (IL-15) is a T cell growth factor which shares manysimilarities with the more ubiquitously known interleukin-2 (IL-2){Steel, 2012, Interleukin-15 biology and its therapeutic implications incancer}. It has a trimeric receptor consisting of an IL-15Rα chain whichis unique to IL-15 and the IL-15β (commonly designated as IL-2/15β)along with common gamma chain (γC) which it shares with IL-2. Both theseinterleukins are involved in T cell growth, expansion, activation andsurvival, but there are specific differences between the two. Mostimportantly, IL-15 does not cause activation induced cell death (AICD)unlike IL-2 {Munger, 1995, Studies evaluating the antitumor activity andtoxicity of interleukin-15, a new T cell growth factor: comparison withinterleukin-2; Marks-Konczalik, 2000, IL-2-induced activation-inducedcell death is inhibited in IL-15 transgenic mice}. IL-15 is required fordifferentiation of NK cells, CD8 cells and maintain memory phenotype CD8cells which is the main defense mechanism against cancer cells{Jakobisiak, 2011, Interleukin 15 as a promising candidate for tumorimmunotherapy}. Its receptor IL-15Rα is involved in high affinitybinding of IL-15 which presents it to IL-2/IL-15βγ which in turn leadsto the signaling mediated through various pathways including JAK1/3 andSTAT3/5 pathways {Budagian, 2006, IL-15/IL-15 receptor biology: a guidedtour through an expanding universe; Okada, 2015, STAT3 signalingcontributes to the high effector activities of interleukin-15-deriveddendritic cells}.

To increase the efficacy of CARTs, extra co-stimulatory and cytokinesignaling domains were incorporated to obtain advanced generation ofCARs, abrogating the need to administer additional cytokines along withthe CART infusion. Based on this approach, a fourth generation CAR wasdeveloped with CD27 co-stimulatory domain in addition to CD28 and 4-1BBsignaling domains and its therapeutic efficacy was tested in leukemiaand lymphoma patients. The study herein reports a novel CAR designincorporating the IL-15Rα-signaling domain and the comparison of the newCAR with both the 3^(rd) (19z, with 4-1BB and CD28 domains) and the4^(th) generation CARs (273z, with 4-1BB, CD27 and CD28 domains). Thestudy herein demonstrates that with the addition of IL-15Rα signaling,the CARTs displayed rapid expansion and target killing activities, whichcould translate into high therapeutic efficacy against B cellmalignancies.

Materials and Methods

Cell lines and culture media. RS4;11 and MV4-11 were CD19 positive acutelymphoblastic leukemia and CD19 negative acute mono-myelocytic leukemiacell lines, respectively, which were obtained from American Type CultureCollection (ATCC). Jurkat T cells were purchased from ATCC. All celllines were maintained in RPMI1640 medium (Life Technologies, Inc., GrandIsland, N.Y.) with 10% fetal bovine serum (FBS, Atlanta Biologicals,Inc. Norcross, Ga.) and supplemented with penicillin (100 units) andstreptomycin (100 μg). Target cancer cell lines were transduced withlentiviral vectors expressing a green fluorescent protein (wasabi GFP)and the reporter gene positive cells were sorted by flow cytometry.Jurkat T cells were transduced with lentiviral CAR vectors and the copynumber of CAR per cell was determined by quantitative PCR using genomicDNA.

Blood donors and primary T cell culture. Buffy coats from anonymoushealthy donors (HDs) were purchased from LifeSouth Civitan Blood Center(Gainesville, Fla., USA). PBMCs were isolated from buffy coats bygradient density centrifugation in Ficoll-Hypaque (GE HealthcareBio-Sciences AB, Piscataway, N.J., USA) as previously described {Chang,1995, Infection and replication of Tat-human immunodeficiency viruses:genetic analyses of LTR and tat mutations in primary and long-term humanlymphoid cells}. Blood and bone marrow samples of children with newlydiagnosed acute lymphoblastic leukemia were obtained with the assistanceof a Hematological Malignancies Bank. T cells were activated usinganti-CD3 and anti-CD28 antibody-conjugated magnetic beads orphytohemagglutinin (PHA). The T cells were maintained in TexMACS medium(Miltenyi Biotec Inc, San Diego, Calif.) supplemented withinterleukin-2, -7 and -15 as previously described {Okada, 2015 #33}.Phenotype of the activated cells was verified to confirm T cell purity.After expansion for two to six days, the T cells were transduced withlentiviral CAR vectors.

Lentivector Construction and CAR Gene Transduction.

Lentivectors were generated using the NHP/TYF lentivector system aspreviously described {Chang, 2005 #181; Wang, 2006 #19}. CAR DNA waschemically synthesized and cloned into pTYF transducing vector behindhuman EF1α promoter. The final lenti-CAR vectors were verified byrestriction enzyme mapping and DNA sequencing.

qPCR Determination of CAR Copies in CARTs.

The CAR transgene copy numbers in CARTs were determined by quantitativeSYBR green real time PCR (qRT-PCR) as previously described {Okada, 2015#33}. Genomic DNA was harvested from CARTs using Promega Wizard genomicDNA purification kit (Promega Corp. Madison, Wis.). Conditions for theqRT-PCR reaction was conducted as suggested by SABioscience using theMX3000P qPCR system (Stratagene, Agilent Technologies, Santa Clara,Calif.).

Calcein AM Labeling of Target Cells.

Fresh or thawed leukemia cells obtained from patients were first washedwith RPMI. Then 1×10⁶ cells were suspended in 1960 of RPMI1640 in anon-attachment tube and 40 of 20 μM Calcein AM (Life Technologies Corp)was added to get a final concentration of 0.4 μM. The cells were thenincubated at 37° C. for 1 hour and subsequently washed twice withRPMI1640 with FBS before using for co-culture experiments.

CART Killing Assay.

Target cells and CARTs were counted using tryptan blue staining and ahemocytometer. The cells were co-cultured in 96 U plate ineffector:target ratios of 1-2:1, or as indicated in each experiment.CART numbers ranged from 5×10⁴ to 2×10⁵. The coculture was incubated inTexMACS medium without cytokines. Cells were incubated for 1-2 hours andovernight for short term assays. CART-targeted killing was recorded byquantitative analysis of the shifted side scattered (SSC) population,and early and late apoptotic (annexin V-stained) and Propidium Iodide(PI)-stained (dead) cells {Zhang, 1997, Early detection of apoptosisusing a fluorescent conjugate of annexin V}. Samples were run in theLSRII flow cytometer using the DiVa software (BD Biosciences) andresults were analyzed using the FlowJo software.

Annexin V and PI Staining for Apoptosis.

After 1 to 2 hours of incubation, cells were transferred to 96-V plateand centrifuged at 400 g for 5 minutes to remove the media. Then thecell pellet was washed with PBS once. Cells were re-suspended in 30 μlof staining mix consisting of 0.25 μl PI (Sigma), 0.5 μl Annexin V (BDBioscience) and 29.25 μl of binding buffer (BD Bioscience) and thenincubated in the dark for 15 min at room temperature. Subsequently,cells are suspended in 200 μl FACS buffer and analyzed in the LSRII flowcytometer using DiVa software (BD Biosciences). Results were evaluatedusing FlowJo software.

CFSE Cell Proliferation Assay.

1×10⁵ CARTs were first washed with PBS and then re-suspended in 100 μlof PBS. A working solution of CFSE was prepared in PBS to get a finalconcentration of 10 μM. Then 100 μl of this was added to the cellsuspension to get a final CFSE concentration of 5 μM. The cells wereincubated at 37 C in the dark for 15 minutes. Then the cells were washedtwice with cold RPMI+10% FBS and again once with Texsmacs medium. Thesecells were co-cultured with RS4;11 in a effector to target ratio of 1:5and incubated in the dark at 37 C. For positive control, CFSE labeled Tcells were stimulated with anti-CD3 and anti-CD28 Abs with cytokinemedium, and for negative control, CFSE labeled T cells were treated withmitomycin C. The cells were analyzed by flow for proliferation indicatedby CFSE dilution on Day 2 or 3 after surface staining for CD3 to gatethe T cells only.

Monoclonal Antibodies (mAbs).

Fluorochrome-conjugated mAbs against human IFN-γ (B27, APC), IL-2(MQ1-17H12, PE) CD8 (SK1, APC-Cy7), CD4 (RPA-T4, PB), CD22 (S-HCL-1,APC), CD27 (M-T271, APC), CD28 (L-293, PerCp-Cy5.5), CD34 (8G12,PE-Cy7), PD1 (EH12.1, PE-Cy7), and CD107a (H4A3, FITC) were purchasedfrom BD Biosciences (San Diego, Calif.). Anti-CD19 mAb (SJ25C1, PE) waspurchased from Caltag laboratories (Life technologies Inc) andanti-CD127 mAb (RDR5, APC-eflour) was purchased from eBiosciences.

Surface Staining and Intracellular Cytokine Staining.

For effector functional analysis, the CARTs were mixed with target inE/T ratio of 1:3 overnight with the addition of 1.5 μl ofFITC-conjugated anti-CD107a Ab. Positive control used T cells (withoutCAR) stimulated with PMA (1 μg/μl) and Ionomysin (1 ng/μl) for 1 hour.The intracellular cytokines were immobilized using monensin (6 μg/μl)for 6 hours. The samples were then washed, blocked with 10% human andmouse sera for 30 min, stained with anti-CD4 and anti-CD8 Abs for 30min, fixed and permeabilized with BD Fix/Perm Buffer, stained withanti-IFN-γ and anti-IL-2 Abs for 1 hour, and then analyzed by flowcytometry. Data was collected on the BD LSRII flow cytometer andanalyzed with Flowjo.

Effector Cytokine Analysis Using Cytokine Bead Array.

The BD CBA™ Human Soluble Protein Flex Set System was used to detectconcentrations of cytokines IL-2, IL-6, TNFα and IFNγ in thesupernatants collected from the CART killing assays on day 1 or 2 ofincubation. The CBA system captures a soluble analyte or set of analyteswith beads of known size and fluorescence, making it possible to detectanalytes using flow cytometry. Each capture bead was coated with acapture antibody specific for a soluble protein. The detection reagentwas a mixture of PE-conjugated Abs, which provided a fluorescent signalin proportion to the amount of bound analyte. First 10 tubes of 50 μlset standard dilutions were prepared. Then 50 μl of each unknown samplewas added to an assay tube. 50 μl of the mixed capture beads was addedto each assay tube and incubated at room temperature for 1 hr. Then 50μl of the PE detection reagent was added and incubated for 2 hr at roomtemperature. 1 ml of wash buffer was added to each tube and centrifugedat 200 g for 5 minutes. After the supernatant was removed, 300 μl ofwash buffer was added and vortexed prior to acquiring the results onflow cytometer.

CAR Gene Sequence:

The amino acid sequence of the IL-15Rα CD19 CAR tested in this study isas follows, broken down by the domains included in the CAR:

(CD19 scFv domain) DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAA(CD28 transmembrane/cytoplasmic domain)IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPR  DFAAYRSAS (linker)GGGGSGGGGS (IL-15Ra cytoplasmic signal domain)KSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL (linker) GGGGSGGGGS(CD3zeta signal domain) (SEQ ID NO: 31)RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR 

Results Construction of CARs Containing Various Co-Stimulatory SignalingDomains

The CD19 CAR (19z) sequence was codon optimized and chemicallysynthesized based on the anti-CD19 scFv of mouse hybridoma FMC63{Nicholson, 1997, Construction and characterization of a functional CD19specific single chain Fv fragment for immunotherapy of B lineageleukaemia and lymphoma}. Briefly, the parental 19z CAR(3^(rd)generation) contained a chimeric intracellular signaling elementderived from CD28 trans-membrane and cytoplasmic domain, theco-stimulatory 4-1BB intracellular TRAF binding domain and the CD3ζchain intracellular domain, analogous to the CAR design of Kochenderferet al. {Kochenderfer, 2009, Construction and preclinical evaluation ofan anti-CD19 chimeric antigen receptor}. To establish new generationCARs, the co-stimulatory domains of various immune modulatory receptorswere incorporated into the CAR construct including the cytoplasmicdomains of CD27 (273z), OX40 (OX40z), ICOS (ICOSz), and IL-15Rα (153z).These CARs were cloned into the lentiviral vector pTYF and packaged withNHP/VSVG lentivector system as illustrated in FIG. 1.

Development of a Simplified Killing Assay for Functional Evaluation ofCARs

To demonstrate CAR-mediated specific killing of target cells, asimplified killing assay was developed based on CAR-modified Jurkat Tcells and target cells that were genetically engineered to express agreen fluorescent protein (wasabi) or labeled with calcein AM. Toillustrate CART-mediated target killing, the Jurkat-CARTs (effectors)were incubated with the GFP-labeled target at different effector:targetratios, and at various time points, the cultured cells were stained withannexin V and PI and analyzed by flow cytometry. The diagram in FIG. 2illustrates the killing assay; representative flow cytometry graphsdemonstrate the ratio change of the green target cells (%), the alteredtarget cell morphology shown by increased side scatter (SSC) reflectingapoptosis, and the populations of early and late apoptotic cells(annexin V-stained) and dead cells (PI-stained). All CAR constructs wereverified for specific target killing activities prior to the primary Tcell gene transfer studies.

Evaluation of CART Killing Efficiency Using Primary T Cells

Jurkat CARTs are convenient for short term killing assessment. However,Jurkat CARTs lack long term killing function due to high PD-1up-regulation after target engagement. Therefore, T cells from healthydonors were transduced with lenti-CAR vectors and tested for CART targetkilling activities. The efficiency of lentivector modification ofprimary T cells is very high (FIG. 3). Healthy donor T cells weretransduced with 19z, 273z OX40z, ICOSz, and 153z lenti-CAR vectors. Ascontrol, GD2-CARTs expressing the ganglioside antigen GD2-specific CARwere included. RS4;11w, a CD19+ve B-ALL cell line expressing wasabi GFPgene, was used as a specific target. Both target and effector cells wereco-cultured in a ratio of 1:2 in a 96 U shaped well plate. Killing wasassessed at 24-48 hr and pictures were obtained under fluorescentmicroscope. Representative results are illustrated in FIG. 4, whichshows almost complete disappearance of the green target cells in the153z coculture well, and decreased target in 19z, 273z, OX40z and ICOSzgroups compared to target only and the non-specific GD2-CART groups.Repeated assays showed consistently that 153z had a rapid killingkinetics compared to the others.

Highly Efficient and Rapid Target Killing Kinetics of 153z CAR in BothHealthy Donor and Leukemic Patient T Cells

To further evaluate the rapid target killing activities of 153z CAR, theRS4;11w target was cocultured with healthy donor PBMC-derived 19z, 273z,153z and control GD2z CARTs at 1:1 ratio for one hour, and cells werestained with annexin V and PI and analyzed by flow cytometry. The resultconfirmed that 153z CARTs killed the target at the highest efficiencyquickly based on the analysis of early apoptotic cells by annexin Vstaining (annexin V positive and PI negative, FIG. 5A). This assay wasrepeated in triplicates and both percentages of total cell death andspecific target lysis were analyzed, and statistical significance wasobtained as shown in FIG. 5B.

To examine the activities of 153z CAR in leukemic patients' T cells,RS4;11wasort target killing assay was performed using CARTs generatedfrom B-ALL patients' T cells. 19z, 273z and 153z CARs were compared inthree leukemia patients' T cells in a short term assay as describedabove. The results again confirmed that 153z displayed the fastestkilling kinetics as illustrated by annexin V staining for earlyapoptotic cells in 1-2 hours (FIG. 6A). This was evident after 1-3 daycoculture when cell death was analyzed by annexin V/PI staining and thedisappearance of GFP+ target cells examined by flow cytometry and underfluorescent microscope (FIGS. 6B, 6C).

Effective Killing of Patient Leukemic Cells by Autologous 153z CARTs

The above assays used B-ALL cell line RS4;11 as target cells. Toillustrate killing of the patients' own leukemic cells, B-ALL patients'leukemic cells were obtained from bone marrow of two patients. Thesecells were labeled with green fluorescent dye calcein AM and co-culturedwith autologous CARTs of 19z, 273z and 153z from the two B-ALL patientsat effector:target ratio of 1:5. Target cell death was examined after 1hour and 1 day. FIG. 7 demonstrates that 153z CARTs displayed the sametrend of high target killing activities as compared with 19z and 273z in1 hr (FIG. 7A) and 1 day (FIG. 7B) co-cultures of patients' own leukemiccells. Similar results were obtained for the second patient (not shown).

Prolonged Killing Function of 153z CARTs

Next it was determined if these CD19 CARTs retained their ability tokill more target cells after they had already been exposed to targetonce. After the green target was completely gone, the remaining livecells were counted, which were mainly the CART effector cells. An equalnumber of the various CARTs was added to 10 times RS4;11w target infurther rounds of killing, and flow cytometry was performed to determinethe green target cells left at various time points. Both a healthy donorand a leukemic patient CARTs were tested. The results showed that targetwas eliminated efficiently after even fourth rounds of coculture, allwithin 1-2 days, as shown with the leukemia patient CARTs in FIG. 8.Importantly, 153z, although killed rapidly, retained capacity to furtherkill more target cells.

Increased T Cell Proliferation and Effector Functions of 153z CAR

The above results indicate that 153z had high target killing activitiescompared to 19z and 273z. Without wishing to be bound by theory, thetarget killing function of the CAR should be correlated withintracellular signaling for T cell proliferation and effector cytokinerelease. Next, T cell proliferation assays were performed based on CFSElabeling of T cells. Control T cells or CARTs were labeled with CFSE andcocultured with leukemic target cells for 3 days. Flow cytometryanalysis revealed that 153z CARTs displayed the highest proliferationrate as compared with 19z and 273z CARTs (FIG. 9). For the effectorfunction analysis, CARTs were stained for intracellular IL-2, IFN-γ anddegranulated CD107a after incubation with target cells. The resultsillustrated that 153z CARTs displayed the highest effector cytokineexpression when co-cultured with target (FIGS. 10A-10C). To confirm theintracellular cytokine results, a flow cytometry-based cytokine beadassay (CBA) was used to determine the concentration of various cytokinessecreted into the cocultures. In this assay, T cells without CAR andwith no target added and T cells stimulated with PMA and ionomysin wereincluded as negative and positive controls, respectively. Supernatantsfrom 153z CART co-culture collected on 48 hr contained the highestamount of IFN-γ and IL-2 compared to 19z and 273z CART co-cultures.

Genetically modified T cells have shown great promise in the treatmentof B cell malignancies in the last five years. Among a number ofpublished CART trials in hematological malignancies, CD19 or CD20 hasbeen an effective target {Porter D L, 2011, Chimeric antigenreceptor-modified T cells in chronic lymphoid leukemia.; Brentjens R J,2013, CD19-targeted T cells rapidly induce molecular remissions inadults with chemotherapy-refractory acute lymphoblastic leukemia.;Kochenderfer J N, 2013, Donor-derived CD19-targeted T cells causeregression of malignancy persisting after allogeneic hematopoietic stemcell transplantation.; Grupp S A, 2013, Chimeric AntigenReceptor-Modified T cells for Acute Lymphoid Leukemia.}. Severalvariables are still being investigated in determining the CART efficacyand persistence in vivo. One of the important determinants of CARTefficacy is the choice of activation or co-stimulatory signals in theCAR structure. The study described herein made an attempt to furtherperfect the CAR design by exploring the potent activating signal ofIL-15Rα (153z CAR) as a co-stimulatory domain and found that these CARTshave rapid reaction kinetics and high targeting efficacy. To assess CARfunctions, an efficient lentiviral vector gene delivery system has beenapplied and a rapid cytotoxic assay without pre-selection of CARtransduced T cells has been developed. The different CARTs weretransduced with lenti-CAR vectors at similar efficiencies based onquantitative PCR confirming the CAR transgene copy number in the cells.The 153z CARTs killed target CD19+ cells very rapidly as compared to19zand 273z CARTs in short term assays from 30 min to two days. To assesslong term killing effects, more than ten times of target cells wererepetitively added to the CARTs, and interestingly, the CARTs becamemore effective after the first round of killing, likely due to selectiveexpansion of functional CARTs. The latter is similar to the findings ofHenderson et al. who has reported enhanced killing of CARTs onre-encountering the target {Henderson, 2013, Chimeric antigenreceptor-redirected T cells display multifunctional capacity andenhanced tumor-specific cytokine secretion upon secondary ligation ofchimeric receptor}. This is also consistent with in vivo observationthat infused CARTs can expand more than 1,000 fold in leukemia patients{Porter D L, 2011, Chimeric antigen receptor-modified T cells in chroniclymphoid leukemia.; Brentjens R J, 2013, CD19-targeted T cells rapidlyinduce molecular remissions in adults with chemotherapy-refractory acutelymphoblastic leukemia.}.

The assessment was further conducted using the primary patient T cellswith CAR and patient ALL cancer cells. A much higher ratio oftarget:effector such as 10:1 was tested to closely parallel the in vivosituation where tumor burden may be much higher compared to the infusedCART cells. Effective killing was observed even in this setting. Theseresults indicate that the CD19-specific CART cells can efficientlytarget ALL coupled with high proliferation rate, activation of effectorfunctions, resulting in complete eradication of cancer cells. TheCFSE-based proliferation assay indicates that the 153z CARTsproliferated to the greatest extent compared to the 19z and 273z CARTs.One of the factors contributing to the enhanced killing seen with 153zCARTs might be the rapid expansion of these cells when they see target.In addition, 153z CARTs secreted increased amount of effector cytokinessuch as IFN-γ, TNFα and IL-2 upon target engagement, as compared with19z and 273z CARTs. Therefore, without wishing to be bound by theory,the enhanced target killing of 153z CARTs is conceivably a result ofboth rapid response kinetics and high effector activities.

As with T cell nature and biology, expansion and differentiation couldresult in exhaustion. 153z cells killed more rapidly and efficientlywhen they encountered target, so they could exhaust rapidly as well.Re-targeting experiments were performed by counting the cells from theco-culture after all the target was gone and adding more target in aratio of 10:1 target to effector to a 48 well plate. This retargetingdemonstrated that the target was killed more rapidly as the T cells werealready in an activated, effector state. The 153z cells retained theircapacity to kill as effectively as the 19z and 273z cells in theretargeting experiment. Interestingly, the increased activated state of153z CART was associated with decreased PD-1 expression on these cellsafter the first round killing, suggesting that initial expansion of 153zCARTs is not very exhaustive.

Without wishing to be bound by theory, when CART binds the tumorantigen, this sends an activation signal and converts the memory T cellsinto effector T cells which mediates the killing by perforin-granzymepathway, and the release of effector cytokines leading to sustained Tcell activation {Trapani, 2002, Functional significance of theperforin/granzyme cell death pathway}. CD107a degranulation is anothermarker for T cell effector function associated with cytolytic activities{Betts, 2004, Detection of T-cell degranulation: CD107a and b. Methodsin Molecular Medicine}. The study herein showed the differentialproduction of these cytokines and up-regulation of CD107a expressionwhen the different CART cells were encountering target by intracellularcytokine staining, and confirmed that these effector markers were inline with the strength of the T cell activation signal and targetkilling function. Again, the highest effector markers were observed with153z CARTs.

In a similar study, Hoyos et al. have reported CARTs with CD28co-stimulatory domain along with IL-15 gene and an inducible caspasegene, which ectopically over express IL-15 to enhance killing, whichcould potentially lead to excessive IL-15 production which is known tobe a tumor growth cytokine {Hoyos, 2010, Engineering CD19-specific Tlymphocytes with interleukin-15 and a suicide gene to enhance theiranti-lymphoma/leukemia effects and safety}. In 153z CAR, the IL-15Rαcytoplasmic domain is engineered in tandem with CD28 and CD3, whichconfers a stimulating effect only when the CARTs encounter target. Thus,this design endows IL-15 signaling in the CARTs only independent ofextracellular IL-15 expression.

In summary, the IL-15Rα endodomain confers a potent stimulus to the CARTwhen it encounters target, leading to enhanced proliferation, effectoractivities and target killing. In clinical settings, during the initialtreatment when disease burden is high, CARTs with rapid killing efficacycould be advantageous and once this effect is seen, maintenance CARTsmay be applied for persistence. Besides leukemia, it is also anticipatedthat the rapid 153z CAR effector kinetics may achieve better therapeuticoutcomes in the more difficult to target tumors such as solid tumors.

Example 2: Demonstration of Safety and Efficacy of Chimeric AntigenReceptor (CAR)-Modified T Cells for the Treatment of Relapsed orRefractory CD30 Positive Lymphomas

The leukocyte activation marker CD30 (TNFRSF8) is a 120 kDa type Itransmembrane cytokine receptor from the tumor necrosis factor receptor(TNFR) superfamily. CD30 is consistently expressed in Hodgkin lymphoma(HL), anaplastic large cell lymphoma (ALCL), and variably expressed inother B and T cell lymphomas, including diffuse large B-cell lymphoma(DLBCL), primary effusion lymphoma, adult T-cell leukemia/lymphoma,mycosis fungoides, and extranodal natural killer/T-cell lymphoma. Alarge number of CD30 positive lymphoma patients cannot be cured bystandard chemo-radiotherapy. Brentuximab Vedotin (SGN-35) is anantibody-drug conjugate directed against the CD30 antigen expressed onlymphoma cells, which has been approved by U.S. Food and DrugAdministration (FDA) for the treatment of relapsed or refractoryclassical HL and systemic ALCL. However, SGN-35 is not available orapproved in many countries. Therefore, although CD30 represents anattractive target for immunotherapy, there remains a need to developalternative CD30-targeting therapeutics.

Described in this example are a experiments related to a chimericantigen receptor (CAR) that targets CD30 and a clinical trial ofautologous T cells expressing this CAR. Pre-clinical study results andthe preliminary outcome of one patient enrolled in a clinical trial ofanti-CD30 CAR T cells for the management of relapsed and refractory CD30positive lymphomas (www.clinicaltrials.gov; #NCT02274584) are reported.

Materials and Methods Cell Lines and Media

CD30-positive target cells were used for the CAR T cell target killingassays. Primary CD30+ ALCL cells and B lymphoma cells were establishedin the laboratory. These cells were maintained in RPMI1640 medium (LifeTechnologies, Inc. Grand Island, N.Y.) supplemented with 10% fetalbovine serum (Atlanta Biologicals, Inc. Norcross, Ga.), penicillin (100units/ml) and streptomycin (100 ug/ml). Target cell lines weretransduced with lentiviral vectors expressing a green fluorescentprotein (wasabi GFP) and the reporter gene positive cells were sorted byflow cytometry (see FIG. 11). T cells were transduced with lentiviralCD30 CAR or control CAR vectors and the copy number of CAR per cell wasdetermined by quantitative PCR using genomic DNA.

Blood Donors and Primary T Cell Culture

Buffy coats from healthy donors (HDs) were purchased from LifeSouthCivitan Blood Center (Gainesville, Fla., USA) with approval ofInstitutional Review Board (IRB-01) of University of Florida. PBMCs wereisolated from buffy coats by gradient density centrifugation inFicoll-Hypaque (GE Healthcare Bio-Sciences AB, Piscataway, N.J., USA).Blood and bone marrow samples of Hodgkin's lymphoma patients wereobtained from Peking University Cancer Hospital with IRB approvedprotocol. T cells were activated using anti-CD3 and anti-CD28 antibodiesor phytohemagglutinin (PHA). The T cells were maintained in TexMACSmedium (Miltenyi Biotec Inc, San Diego, Calif.) supplemented withinterleukin-2, -7 and -15. Phenotype analysis of the activated cells byflow cytometry was performed to confirm T cell purity. After expansionfor two to six days, the T cells were transduced with lentiviral CARvectors.

CD30 CAR Synthesis, Lentiviral Vector Construction and CAR GeneTransduction

The CD30 CAR sequence was codon optimized and chemically synthesizedbased on two published mAb clones, AC10 (a murine hybridoma IgG2b clone,J. Immunol. 1993, Dec. 1; 151(11): 5896-5906), and a humanized mAb clone5F11. The amino acid sequences of the two scFv clones used for CD30 CARengineering are listed below.

AC10 scFv: VH (CDR1) QIQLVQSGAEVKKPGASVKVSCKAS GYTFTDYYIT  (CDR2)WVRQAPGQGLEWMG WIYPGSGNTKYNEKFKG (CDR3) RVTMTRDTSISTAYMELSRLRSDDTAVYYCANYGNYWFAY (SEQ ID NO: 14) WGQG TLVTVSS  VL: (CDR1)DIVMTQSPDSLAVSLGERATINC KASQSVDFDGDSYMN (CDR2) WYQQKPGQPPKLLIY AASNLES(CDR3) GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQSNEDPWT (SEQ ID NO: 13)FGQGTKVEIK 5F11 CD30 scFv: VH (CDR1) QVQLQQWGAGLLKPSETLSLTCAVYGGSFSAYYWS (CDR2) WIRQPPGKGLEWIG DINHGGGTNYNPSLKS (CDR3)RVTISVDTSKNQFSLKLNSVTAADTAVYYCAS LTAY  (SEQ ID NO: 16) WGQGSLVTVSS VL(CDR1) DIQMTQSPTSLSASVGDRVTITC RASQGISSWLT  (CDR2) WYQQKPEKAPKSLIYAASSLQS (CDR3) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQYDSYPIT(SEQ ID NO: 15) FGQGTRLEIK

The CAR contains a chimeric intracellular signaling element derived fromCD28 trans-membrane and cytoplasmic domain, the co-stimulatory 4-1BBintracellular TRAF binding domain, a CD27 cytoplasmic domain, and theCD3ζ chain intracellular domain. The CAR gene was cloned into thelentiviral vector pTYF and packaged with NHP/VSVG lentivector system, asshown in FIG. 12.

Lentiviral vectors were generated based on the NHP/TYF lentiviral vectorsystem. CAR DNA was chemically synthesized and cloned into pTYFtransducing vector behind human EF1α promoter. The final lentiviral-CARconstructs were verified by restriction enzyme mapping and DNAsequencing.

CAR Gene Sequence

The amino acid sequence of the CD30 CAR tested in this study is shown asfollows, broken down by the domains included in the CAR. A schematic ofa CD30 CAR is shown in FIG. 13:

(CD30 scFv domain) QVQLQQWGAGLLKPSETLSLTCAVYGGSFSAYYWSWIRQPPGKGLEWIGDINHGGGTNYNPSLKSRVTISVDTSKNQFSLKLNSVTAADTAVYYCASL TAYWGQGSLVTVSS(Linker) GSTSGSGKPGSSEGSTKG (CD30 scFv domain)DIQMTQSPTSLSASVGDRVTITCRASQGISSWLTWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDSYPITF GQGTRLEIK (Linker)GSTSGSGKPGSSEGSTKG (CD28 transmembrane domain)FWVLVVVGGVLACYSLLVTVAFIIFWV (4-1BB intracellular domain)KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL  (CD27 intracellular domain)QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP (Linker)GSTSGSGKPGSSEGSTKG  (CD3 zeta domain)RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPR (Linker) GSTSGSGKPGSSEGSTKG  (truncated iCasp9 domain)VGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (FKBP domain) (SEQ ID NO: 32)MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHA  TLVFDVELLKLEIn some embodiments, the above exemplary, non-limiting arrangements arefrom left to right, N-terminus to C-terminus of the CAR. The CAR maycomprise or further comprise any other combination of elements asdescribed herein.

CAR Detection by PCR

The CAR transgene copy numbers in CART cells were determined byquantitative SYBR green real time PCR (qRT-PCR). Genomic DNA washarvested from CART cells using Promega Wizard genomic DNA purificationkit (Promega Corp. Madison, Wis.). The qRT-PCR reaction condition was assuggested by SABioscience and data collected using MX3000P (Stratagene,Agilent Technologies, Santa Clara, Calif.).

Calcein AM Labeling of Primary Tumor Cells

Fresh or thawed tumor cells were first washed with RPMI1640 medium. Then1×10⁶ cells were suspended in 1960 of RPMI1640 in a tube, and 4 μl ofCalcein AM (Life Technologies Corp., 20 μM) was added to get a finalconcentration of 0.4 μM. The cells were incubated at 37° C. for 1 hourand washed twice with RPMI containing fetal bovine serum before used inco-culture experiments.

CAR T Cell Killing Assay

Target cells and CAR T cells were counted using trypan blue staining anda hemocytometer. The cells were co-cultured in 96 U plate ineffector:target ratios of 1-2:1, or as indicated in each experiment. CART cell numbers ranged from 5×10⁴ to 2×10⁵. The coculture was incubatedin TexMACS medium without cytokines. Cells were incubated for 1-2 hoursor overnight for short term assays. CAR T-targeted killing was recordedby quantitative analysis of the shifted side scattered (SSC) population,and early and late apoptotic (annexin V-stained) and Propidium Iodide(PI)-stained (dead) cells. Samples were run in the LSRII flow cytometerusing the DiVa software (BD Biosciences) and results were analyzed usingthe FlowJo software. An example of results obtained using this assay isshown in FIG. 14.

Annexin V and PI Staining for Apoptosis

At different time points after incubation, cells were transferred to96-V plate and centrifuged at 400 g for 5 minutes to remove the media.Then the cell pellet was washed with PBS once. Cells were re-suspendedin 30 of μl staining mix consisting of 0.25 μl PI (Sigma), annexin V (BDBioscience) and 29.25 μl of binding buffer (BD Bioscience), and thenincubated in the dark for 15 minutes at room temperature. Subsequently,cells were suspended in 200 μl FACS buffer and analyzed in the LSRIIflow cytometer using DiVa software (BD Biosciences). Results wereevaluated using FlowJo software.

Monoclonal Antibodies

Fluorochrome-conjugated monoclonal antibodies against human IFN-γ (cloneB27, APC), IL-2 (clone MQ1-17H12, PE) CD8 (clone SK1, APC-Cy7), CD4(clone RPA-T4, PB), CD30 (clone 50614, FITC), CD27 (clone M-T271, APC),CD 28 (clone L-293, PerCp-Cy5.5), CD34 (clone 8G12, PE-Cy7), PD1 (cloneEH12.1, PE-Cy7), CD107a (clone H4A3, FITC) were purchased from BDBiosciences (San Diego, Calif.). Anti-CD19 (clone SJ25C1, PE) andanti-CD127 (clone eBio-RDR5, APC-eflour) antibodies were purchased fromCaltag Laboratories (Life technologies Inc) and eBiosciences,respectively.

Surface Staining and Intracellular Cytokine Staining of CAR T Cells

For effector functional analysis, the CART cells were mixed with targetcells in an E:T ratio of 1:3 overnight with the addition of 1.5 μl ofFITC-conjugated anti-CD107a Ab. Positive control used T cells (withoutCAR) stimulated with PMA (1 μg/μl) and Ionomysin (1 ng/μl) for 1 hour.The intracellular cytokines were immobilized after treated with monensin(6 μg/μl) for 6 hours. The samples were then washed, blocked with 10%human and mouse sera for 30 min, stained with anti-CD4 and anti-CD8 Absfor 30 min, fixed and permeabilized with BD Fix/Perm Buffer, stainedwith anti-IFN-γ and anti-IL-2 Abs for 1 hour, and then analyzed by flowcytometry. Data was collected on the BD LSRII flow cytometer andanalyzed with FlowJo.

Results

The patient is a 22 year-old male. He was diagnosed with stage IIIHodgkin's lymphoma (Nodular Sclerosis), and was treated with six cyclesof ABVD and radiation to the residual disease. The response to thistreatment was complete remission. Subsequently, the disease relapsedconfirmed by biopsy of the left supraclavicular lymphadenopathy. Aftertwo cycles of COEP, disease kept progressing. The patient then receivedtwo cycles of ICE followed by partial remission. The patient was latertreated with auto-transplant. This treatment resulted in completeremission. The disease relapsed again, with nodules in lung andmediastinal, abdominal, retroperitoneal lymphadenopathy. Subsequently,the patient was enrolled in a clinical trial of anti-CD30 chimericantigen receptor-modified T cells.

Detection of CAR T Cells In Vitro

FIG. 15 shows a fluorescent image of target killing assay of twodifferent CD30-CAR T cells based on 5F11 and AC10 scFv CD30-CARs. Theprimary lymphoma cell line was labeled with green fluorescent proteinand used as target cells. CAR T cells and target cells were coincubatedin a 96 U shaped well at 1:1 ratio for 12 days. The disappearance ofgreen fluorescent indicates target cell killing by CAR T cells. The AC10CD30-CAR displayed the best target killing activity.

FIG. 16 shows flow cytometry target killing assay of two differentCD30-CAR T cells based on 5F11 and AC10 scFv CD30-CARs. The primarylymphoma cell line was labeled with green fluorescent protein and usedas target cells. CAR T cells and target cells were coincubated in a 96 Ushaped well at 1:1 ratio for 12 days. The disappearance of greenfluorescent cells detected by flow cytometry indicates target cellkilling by CAR T cells. The AC10 CD30-CAR displayed the best targetkilling activity.

Detection of CAR T Cells In Vivo

Quantitative polymerase chain reaction was performed to detect CAR Tcells in blood. The CD30-CAR T cells were detected on day 14 after firstinfusion. On day 45, CAR T cells reached its peak level accounting formore than 20% of circulating lymphocytes. This peak level of CAR T cellscoincided with the observed disease remission and results of serumcytokine levels. CAR T cells decreased 80 days after cell infusion,which paralleled with disease progression. Interestingly, CAR T cellsincreased to ˜8% again on day 140 indicating in vivo response to tumorrelapse (FIG. 17).

Detection of Cytokine Response after CAR T Infusion

Serum cytokines were measured and showed evidence of immune activation.The patient's disease remission was accompanied by an increase in levelsof inflammatory cytokines, with levels of interferon-γ and interleukin-6peaking around 40 days after first cell infusion. The increase in serumcytokines paralleled with disease remission (FIGS. 18-19).

Example 3. Chimeric Antigen Receptor Expressing T Cells Mediate ActivityAgainst Osteosarcomas

Since the prognosis for children with high risk osteosarcoma (OS)remains suboptimal despite intensive multi-modality therapies, there isa clear and urgent need for the development of targeted therapeuticsagainst these refractory malignancies. Chimeric antigen receptor (CAR)modified T cells can meet this need by utilizing the immune system'ssurveillance capacity and potent cytotoxic mechanisms against tumorspecific antigen targets with exquisite specificity. Since OS highlyexpresses the GD2 antigen, a viable immunotherapeutic target, the studydescribed herein sought to assess if CAR modified T cells targeting GD2could induce cytotoxicity against OS tumor cell lines. It wasdemonstrated that the OS cell lines U20S, HOS, and a primary human OScell line highly express the GD2 antigen (>80%), and that GD2 CARmodified T cells were highly efficacious for inducing tumor cell death.Interestingly, the OS cells were induced to up-regulate expression ofPD-L1 upon interaction with GD2 CAR modified T cells, and the specificinteraction induced CAR T cells to overexpress the exhaustion markerPD-1 along with increased CAR T cell apoptosis. To further potentiateCAR T cell killing activity against OS, it was demonstrated thatcheckpoint blockade along the PD-L1/PD-1 axis can synergize with CAR Tcell therapy. In addition, conventional chemotherapy in combination withCAR T cell therapy can also synergize the effects showing an increasedtarget cell killing activities.

Materials and Methods Cell Lines

HOS and U2OS cells were cultured at 37° C. in 4% CO2 atmosphere inDulbecco's modified eagle medium; DMEM (GIBCO®, Life technologies, CA,USA) supplemented with 10% fetal bovine serum, 1% penicillin and 1%streptomycin.

Jurkat cells were cultured at 37° C. in 4% CO2 atmosphere in RPMI-1640medium (GIBCO, Life Technologies) supplemented with 10% fetal bovineserum, 1% penicillin and 1% streptomycin.

Detection of GD2 Surface Expression

The ˜80% confluent cells were harvested using 2.5 mM EDTA for HOS and 5mM EDTA for U2OS. The cells were sampled, mixed with trypan blue andcounted. For GD2 surface staining, 10⁵ cells were collected and washedtwice with PBS and blocked with 10% human-mouse (1:1) serum in FACSbuffer (2% FBS, 0.1% NaN3 in PBS) at 4° C. for 30 minutes. The cellswere washed with FACS buffer and stained with PE conjugated anti-GD2 Ab(BD biosciences, CA, USA) and kept in the dark at 4° C. for 30 minutes.The cells were washed with FACS buffer and fixed with 1%paraformaldehyde in PBS. GD2 surface expression were detected by BDLSRII flow cytometer and analyzed with FlowJo. Percentage of GD2expression and MFI were determined by subtraction background obtainedfrom isotype control (PE conjugated mouse IgG2a, BD Pharmingen, CA,USA).

Construction of Lentiviral Vectors

Lentiviral vectors were generated using the NHP/TYF lentiviral vectorsystem as previously described [16, 17]. CAR DNA was chemicallysynthesized and cloned into pTYF transducing vector behind human EF1αpromoter using standard molecular cloning approaches. The finallentiviral-constructs were verified by restriction enzyme mapping andDNA sequencing.

Blood Donors, PBMC Isolation and T Cell Activation

Blood samples were obtained from a donation center or osteosarcomapatients. PBMC were isolated using Ficoll-Paque plus (GE Healthcare). Tcells were activated using anti-CD3 and anti-CD28 antibody-conjugatedmagnetic beads or phytohemagglutinin (PHA). The T cells were maintainedin TexMACS (Miltenyi Biotec Inc, San Diego, Calif.) or AIM-V(Invitrogen) supplemented with interleukin-2, -7 and -15 as previouslydescribed [18]. Phenotype analysis of the activated cells were verifiedto confirm T cell purity. After expansion for two to six days, the Tcells were transduced with lentiviral CAR vectors and evaluated killingfunction.

GD2-CAR-T Cell Cytotoxicity Analysis

GFP-positive OS cells were prepared as single cell suspension asmentioned above. The cells were resuspended in DMEM growth medium at1.5×10⁵ cells/ml and 200 μl of the OS cells were seeded into 48-wellplate and cultured at 37° C. in 5% CO2 for two hours. CAR-modified Tcells (primary T cells or Jurkat cells) were added to the wells of OScells at various effecter to target (E:T) ratio and co-cultured forvarious period of time in the present or absent of anti-PD-L1 antibody(BioLegend, CA, USA). The cells were monitored periodically underfluorescence microscope (Zeiss Axiovert 25) and photographed. For flowcytometer analysis of cell death, the cells were harvested using EDTA asdescribed. The harvested cells were washed once with PBS and stainedwith Annexin V-V450 (BD Biosciences) and propidium iodide (Sigma) for 10minutes at room temperature. After staining, the cells were resuspendedin 1% paraformaldehyde in PBS. Data were collected by BD LSRII flowcytometer and analyzed with FlowJo. The difference of cell death betweenGD2 CAR-modified T cells and control-CAR T cells was analyzed byindependent t-test with P value ≤0.05 being significant. Percentspecific lysis of target cells was calculated based on the followingformulation [19]:

% specific lysis=(% apoptosis of target cell−% spontaneous cellapoptosis)/(100%−% spontaneous cell apoptosis)×100

PD-1 and PD-L1 Surface Staining of the Co-Cultured Cells

The co-cultured OS cells and CAR-modified T cells were harvested andblocked with 10% human-mouse (1:1) serum at 4° C. for 30 minutes. Thecells were washed and stained with PE-Cy7 conjugated anti-PD-L1(eBiosciences, CA, USA) or PE-Cy7 conjugated anti-PD-1 (BD Biosciences)at 4° C. for 30 minutes in the dark. The cells were washed with FACSbuffer and fixed with 1% paraformaldehyde in PBS. PD-1 or PD-L1 surfaceexpression was analyzed using BD LSRII flow cytometer and FlowJosoftware. For analysis of PD-1 expression in CAR-modified T cells, theFITC negative T cells were gated while for PD-L1 expression the FITCpositive cells were gated. Percentage of PD-1 or PD-L1 expression andmean fluorescence index (MFI) were determined by subtraction ofbackground isotype control (PE-Cy7 conjugated mouse IgG1K; BDBiosciences).

Results Analysis of GD2 Surface Expression in OS Cell Lines.

Two OS cell lines including HOS and U2OS cells were analyzed forexpression of GD2 by flow cytometry. GD2 expression was found on both OScell lines, 80.1% and 99% on U2OS and HOS cells, with MFI of 7,066 and26,796, respectively (FIG. 20). In a primary OS cell culture, OS156, ahigh level of GD2 expression was detected (94.3%, MFI=72,080, FIG. 20).

Construction of 4th Generation GD2 CAR Lentiviral Vectors.

To engineer GD2-specific CARs, three humanized GD2-specific scFv clones,hu3F8, c.60C3 and hu14.18[15, 20, 21] were selected. These GD2 CARsequences were then human codon-optimized and chemically synthesized. Toestablish 4th generation CARs, several intracellular T cell signalingmotifs were incorporated in the CARs including CD28 transmembrane andcytoplasmic domain, the co-stimulatory 4-1BB intracellular TRAF bindingdomain, the CD27 cytoplasmic domain, and the CD3ζ chain intracellulardomain as illustrated in FIG. 21. These CAR genes were cloned into thelentiviral vector pTYF and packaged into lentiviral particles for genetransfer.

hu3F8 scFv: (VH and VL linked by 218S  linker which is underlined)(SEQ ID NO: 19) QVQLVESGPGVVQPGRSLRISCAVSGFSVTNYGVHWVRQPPGKGLEWLGVIWAGGITNYNSAFMSRLTISKDNSKNTVYLQMNSLRAEDTAMYYCASRGGHYGYALDYWGQGTLVTVSSGSTSGSGKPGSSEGSTKGEIVMTQTPATLSVSAGERVTITCKASQSVSNDVTWYQQKPGQAPRLLIYSASNRYSGVPARFSGSGYGTEFTFTISSVQSEDFAVYFCQQDYSSFGQGTKLEIKC60c3 ScFv: (VH and VL linked by 218S  linker which is underlined(SEQ ID NO: 20) EVKLVESGGGLVLPGDSLRLSCATSEFTFTDYYMTWVRQPPRKALEWLGFIRNRANGYTTEYNPSVKGRFTISRDNSQSILYLQMNTLRTEDSATYYCARVSNWAFDYWGQGTTLTVSSGSTSGSGKPGSSEGSTKGDVVMTQTPLSLPVSLGDQASISCRSSQSLLKNNGNTFLHWYLQKSGQSPKLLIYKVSNRLSGVPDRFSGSGSGTYFTLKISRVEAEDLGVYFCSQSTHIPYTFGGGTK LEIKHu14.18 scFv: (VH and VL linked by 218S linker  which is underlined(SEQ ID NO: 21) EVQLVQSGAEVEKPGASVKISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSTSTAYMHLKSLRSEDTAVYYCVSGMEYWGQGTSVTVSSGSTSGSGKPGSSEGSTKGDVVMTQTPLSLPVTPGEPASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKThe above scFv domains were linked to the following CAR-induciblecaspase 9 sequence (domain names are listed after each domain in bold);

(CD 28 transmembrane domain) FWVLVVVGGVLACYSLLVTVAFIIFWV(4-1BB intracellular domain) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(CD27 intracellular domain)QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP (218S linker)GSTSGSGKPGSSEGSTKG (CD3zeta)RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPR (218S linker) GSTSGSGKPGSSEGSTKG  (truncated Casp9)VGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (FKBP domain) (SEQ ID NO: 37)MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHA TLVFDVELLKLE

Functional Evaluation of GD2-CARs Based on a Rapid Killing Assay.

A simple and quick killing assay was developed to demonstrateGD2-CAR-mediated specific killing of OS cells. The assay was based onco-incubation of CAR-modified T cells and green fluorescent (wasabi GFP)GD2-positive OS cells. CAR T cells (effectors) were incubated with greentarget cells at different effector/target ratios, and at various timepoints, the cultured cells were analyzed by flow cytometry afterstaining with annexin V and propidium iodide (PI). FIG. 22 demonstratesthe killing assay; the target cell death is detected by disappearance ofgreen cells shown by change in E/T ratios and increased apoptotic andPI-stained dead cells. The hu3F8 CAR was chosen as the focus for thefollowing experiments.

GD2-CAR-T Cell Cytotoxicity Against OS Cell Lines.

To demonstrate specific OS killing, the target U2OS and HOS cells weretransduced with a lentiviral green fluorescence wasabi gene to generateU2OSw and HOSw reporter cell lines. The percent specific lysis wasassessed for early and late apoptosis by flow cytometric analysis ofAnnexin V and PI. In a simplified assay, JK cells were transduced withGD2-CAR or control CD19-CAR and cocultured with U2OSw or HOSw to assessthe CAR-T killing ability. Compared with control (non-specific)CD19-CAR-JK cell, the percent specific lysis by GD2 CAR-JK cells wassignificantly increased against U2OSw (FIG. 23A, B; p=0.0049). Similartrend was observed against HOSw (FIGS. 23C, 23D, p=0.078).

Primary GD2 CAR T Cells Targeting Established and Primary OS Cell Lines.

Primary T cells from healthy donors were transduced with GD2 CAR andco-cultured with OS cell lines U2OSw and HOSw in a series of effecter totarget ratios (4:1, 2:1, 1:1 and 1:2), and the cells were harvested andevaluated after 1 day. Compared with control T cells and/or non-specificCAR (CD19-CAR), percent specific lysis of U2OSw was increased whenco-cultured with GD2 CAR T cells but not with non-specific controlCD19-CAR T cells (FIGS. 24A, 24B). Similar results were obtained withHOSw cells using GD2-CAR T cells but not another non-specific glypican 3(GPC3)-CAR T cells (FIGS. 24C, 24D). To see if the OS target killingeffects could be detected with primary tumor cell line, the OS156 cells,established from an OS patient, was tested for GD2-CAR T killing assay.A green fluorescent wasabi gene modified OS156 cell line, OS156w, wasgenerated and used as target cells. After 1 day co-cultured at E:T ratio1:1, GD2 CAR-T cells also efficiently killed the primary OS156w cells asillustrated by specific lysis of target cells and decreased greenfluorescent target cells (FIGS. 24E, 24F).

To see if the GD2-CAR T cells could kill target tumors for extendedperiod, after the first coculture when all tumors cells were killed,more tumor cells were added to the GD2-CAR T cells to set up a secondround killing, at E:T ratio=1:3. Evident under fluorescent microscope,the results showed that GD2 CAR-T cells, but not control T cells ornon-specific CD19-CAR T cells, were able to killed HOSw cells even withthe increased tumor ratio after 12 days in culture (FIG. 24G).

Specific Induction of PD-L-1 on OS Cells and PD-1 on GD2-CAR T.

PD-L1 expression is a key immune evasion mechanism of tumor cells[22].Flow cytometry histogram overlays revealed basal expression of PD-L1 onsurface of both HOS and U2OS cells (FIGS. 25A,25B). Upon specificinteraction with GD2 CAR T cells, up-regulation of PD-L1 in theco-cultured OS cells was observed as illustrated by increased MFI forPD-L1 signal, as compared with the cells co-cultured with control Tcells and CD19 CAR-T cells (FIGS. 25C,25D).

PD-L1 Functions as a Ligand of PD-1 Related to Immune CheckpointInhibition of T Cells Functions.

PD-1 expression was next examined on primary CAR T cells. The resultshowed that primary GD2 CART cells displayed increased PD-1 expressionwhen compared to control T cells and non-specific CD19-CAR T cell after1-day co-cultured with U2OSs and HOSw cells (FIGS. 26A,26B,26C, and26D,26E,26F, respectively, p<0.05).

Example 4. Disialoganglioside (GD2)-Specific Chimeric Antigen Receptor TCells has a High Potent Anti-Tumor Activity on Retinoblastoma Cells

Retinoblastoma (Rb) is an aggressive eye cancer that is the most commonmalignant in infant and children. Decreased doses of chemotherapeutictreatments in infant are considered, however, Rb tumors trend toincrease resistant to the drug. One of the alternative approaches isimmunotherapy based on using chimeric antigen receptor (CAR)-engineeredT cells targeting tumor-specific antigens that are highly expressed onRb tumor, but are present at low levels or not at all in normal tissues.Disialoganglioside 2 (GD2) protein might be a promising target antigen.Therefore, this study aims to investigate the killing efficiency ofGD2-specific CAR T cells to eradicate Rb tumor cells in vitro.

Materials and Methods Cell Culture and Reagents

The human retinoblastoma cell line Y79 RB cells were cultured insuspension in RPMI-1640 medium (Gibco® Life Technologies, USA)supplemented with 15% (v/v) fetal bovine serum (FBS) and 1%Penicillin-Streptomycin solution, and maintained in a 37° C. incubatorwith 5% CO₂. Cytokines recombinant human interleukin (IL)-2, IL-7, andIL-15 (μg/ml) were purchased from a commercial vendor. AnnexinV-PE andpropidium iodide (PI) were purchased from BD Biosciences (BD, USA).Carboplatin was purchased from Sigma.

Generation of GD2 Specific Chimeric Antigen Receptor LentiviralConstructs

To engineer GD2-specific CARs, the humanized GD2-specific scFv clone,hu3F8 [15] was selected. The GD2 CAR sequence were then humancodon-optimized and chemically synthesized. To establish 4th generationCARs, several intracellular T cell signaling motifs were incorporated inthe CAR including CD28 transmembrane and cytoplasmic domain, theco-stimulatory 4-1BB intracellular TRAF binding domain, the CD27cytoplasmic domain, and the CD3 chain intracellular domain asillustrated in FIG. 12. The CAR gene was cloned into the lentiviralvector pTYF and packaged into lentiviral particles for gene transfer.

hu3F8 scFv: (VH and VL linked by 218S linker  which is underlined)(SEQ ID NO: 19) QVQLVESGPGVVQPGRSLRISCAVSGFSVTNYGVHWVRQPPGKGLEWLGVIWAGGITNYNSAFMSRLTISKDNSKNTVYLQMNSLRAEDTAMYYCASRGGHYGYALDYWGQGTLVTVSSGSTSGSGKPGSSEGSTKGEIVMTQTPATLSVSAGERVTITCKASQSVSNDVTWYQQKPGQAPRLLIYSASNRYSGVPARFSGSGYGTEFTFTISSVQSEDFAVYFCQQDYSSFGQGTKLEIKThe above scFv domain was linked to the following CAR-inducible caspase9 sequence (domain names are listed after each domain in bold);

(CD 28 transmembrane domain) FWVLVVVGGVLACYSLLVTVAFIIFWV(4-1BB intracellular domain) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (CD27 intracellular domain)QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP (218S linker)GSTSGSGKPGSSEGSTKG  (CD3zeta)RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPR (218S linker) GSTSGSGKPGSSEGSTKG  (truncated Casp9)VGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (FKBP domain) (SEQ ID NO: 37)MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHA TLVFDVELLKLE

Retinoblastoma Cell Line and T Cells

Y79RB, a GD2 positive retinoblastoma cell line, and Jurkat T cell linewere obtained from American Type Culture Collection (ATCC). These celllines were maintained in RPMI1640 medium (Life Technologies, Inc. GrandIsland, N.Y.) supplemented with 10% fetal bovine serum (AtlantaBiologicals, Inc. Norcross, Ga.), penicillin (100 units/ml) andstreptomycin (100 ug/ml). Target cancer cell lines were transduced withlentiviral vectors expressing a green fluorescent protein (wasabi GFPgene) and the reporter gene positive cells were sorted by flow cytometryor selected with puromycin. Jurkat T cells were transduced withlentiviral CAR vectors and the copy number of CAR per cell wasdetermined by quantitative PCR using genomic DNA.

Blood Donors and Primary T Cell Culture

Buffy coats from healthy donors (HDs) were purchased from LifeSouthCivitan Blood Center (Gainesville, Fla., USA) with approval of anInstitutional Review Board. PBMCs were isolated from buffy coats bygradient density centrifugation in Ficoll-Hypaque (GE HealthcareBio-Sciences AB, Piscataway, N.J., USA) as previously described [21]. Tcells were activated using anti-CD3 and anti-CD28 antibodies. The Tcells were maintained in TexMACS medium (Miltenyi Biotec Inc, San Diego,Calif.) supplemented with interleukin-2, -7 and -15 as previouslydescribed²⁰. Phenotype analysis of the activated cells by flow cytometrywas performed to confirm T cell purity. After expansion for two to sixdays, the T cells were transduced with lentiviral CAR vectors.

Lentiviral Vector Construction and CAR Gene Transduction

Lentiviral vectors were generated based on the NHP/TYF lentiviral vectorsystem as previously described [22,23]. CAR DNA was chemicallysynthesized and cloned into pTYF transducing vector behind human EF1αpromoter. The final lentiviral-CAR constructs were verified byrestriction enzyme mapping and DNA sequencing.

CAR Detection by PCR

The CAR transgene copy numbers in CART cells were determined byquantitative SYBR green real time PCR (qRT-PCR) as previously described[20]. Genomic DNA was harvested from CART cells using Promega Wizardgenomic DNA purification kit (Promega Corp. Madison, Wis.). The qRT-PCRreaction condition was as suggested by SABioscience and data collectedusing MX3000P (Stratagene, Agilent Technologies, Santa Clara, Calif.).

Generation of GD2-Specific Chimeric Antigen Receptor (CAR) T-Cells

Peripheral blood mononuclear cells (PBMCs) were isolated byFicoll-Hypaque density gradient centrifugation, from healthyindividuals. Lymphocytes were stimulated by phytohemagglutinin (PHA)treatment for 2-3 days and maintained in TexMACS™ medium (MiltenyiBiotec, CA, USA). Activated lymphocytes were transduced with CD19- orGD2-specific lentiviral particles and maintained in TexMACS™ mediumsupplemented with human IL-2 (40 U/ml), IL-7 (20 U/ml), and IL-15 (10U/ml) for 3-4 days.

Immunohistochemistry

Tumor samples from RB patients were fixed and embedded in paraffinblocks.

Tumor Cell Cytolysis Assay

Y79 RB wasort cells (target (T) cells) were co-cultured with effector(E) T cells at various effector:target ratios (E:T) in the wells of96-well flat-bottom plate at 37° c. for 24 hours. Target cell lysis wasmonitored under a fluorescence microscope and the cells were collected.Cell death was analyzed using AnnexinV/PI staining by flow cytometry.Specific cell lysis was calculated as [(apoptosis of target cells inco-culture−spontaneous target cells apoptosis)/(100−spontaneous targetcells apoptosis)]×100. Spontaneous target cell death was considered asthe percentage target cell death that cultured in T cell media withouteffector T cells. The results were representative of three independentexperiments.

Flow cytometric Analysis

To determine the expression of GD2 on the surface of primaryneuroblastoma (NB8858) cells and Y79 RB cells, the cells were stainedwith PE-conjugated mouse anti-human GD2 mAb clone 14.G2a (BDBiosciences, CA, USA). Antibody staining was monitored with a BD LSRIIflow cytometer. Data analysis was carried out using FlowJo software(Tree Star Inc., Ashland, Oreg.). For PD-L1 expression, cells werestained with anti-human CD274 or PD-L1 conjugated with PE-Cyanine7monoclonal antibody clone MIHI (eBiosciences, CA, USA).

AnnexinV/PI Staining

After co-culture experiment, cell culture samples were collected foranalyzing tumor cells apoptosis by AnnexinV-PE/PI staining. The cellswere washed and stained with AnnexinV-PE and PI (10 μg/ml) according tomanufacturer's instructions (BD Biosciences). The stained cells wereanalyzed by using flow cytometry on a LSRII. Every group was tested intriplicate.

Early and late apoptotic cells were defined in the population ofAnnexinV+/PI−, and AnnexinV+/PI+ cells, respectively, while the necroticcells were stained with PI+ only. Percent of cell death includes earlyapoptosis, late apoptosis, and necrotic cells. Flow cytometry data wasanalyzed by using FlowJo software (Tree Star Inc., OR)

Surface Staining and Intracellular Cytokine Staining of CART Cells

For effector functional analysis, the CART cells were mixed with targetcells in an E/:T ratio of 1:1 overnight with the addition of 1.5 μl ofFITC-conjugated anti-CD107a Ab. Positive control used T cells (withoutCAR) stimulated with PMA (1 μg/μl) and Ionomysin (1 ng/μl) for 1 hour.The intracellular cytokines were immobilized after treated with monensin(6 μg/μl) for 6 hours. The samples were then washed, blocked with 10%human and mouse sera for 30 min, stained with anti-CD4 and anti-CD8 Absfor 30 min, fixed and permeabilized with BD Fix/Perm Buffer, stainedwith anti-IFN-γ and anti-IL-2 Abs for 1 hour, and then analyzed by flowcytometry. Data was collected on the BD LSRII flow cytometer andanalyzed with Flowjo.

Effector Cytokine Analysis Using Cytokine Bead Array (CBA)

BD CBA™ Human Soluble Protein Flex Set System was used to detectconcentrations of cytokines IL-2, IL-6, TNFα and IFN-γ in thesupernatants collected from the CART cell killing assays on day 1 or 2of incubation. The CBA system captures a soluble analyte or set ofanalytes with beads of known size and fluorescence, making it possibleto detect analytes using flow cytometry. Each capture bead is coatedwith an antibody specific for a soluble protein. The detection reagentis a mixture of PE-conjugated antibodies, which provide a fluorescentsignal in proportion to the amount of bound analyte. First, 10 tubes of50 μl set standard dilutions were prepared. Then 50 μl of each unknownsample was added to an assay tube. 50 μl of the mixed capture beads wasadded to each assay tube and incubated at room temperature for 1 hr.Then 50 μl of the PE detection reagent was added and incubated for 2 hrat room temperature. 1 ml of wash buffer was added to each tube andcentrifuged at 200 g for 5 minutes. After the supernatant was removed,300 μl of wash buffer was added and vortexed prior to acquiring theresults on flow cytometer.

Statistical Analysis

All data are presented as mean±SD. The significance of the differencebetween groups was evaluated by Unpaired Student t-test using Prismsoftware (GraphPad, La Jolla, Calif.). A P value of less than 0.05 wasconsidered statistically significant.

Results GD2 Protein is Highly Expressed in Retinoblastoma Tumor Samplesand Y79 RB Cell Line

GD2 is a ganglioside protein that is highly expressed on the cellsurface. Retinal tissue sample from eight Rb patients were examined forGD2 expression by using immunohistochemistry. Paraffin-embedded tissueswere immunostained with anti-human GD2 antibody and detected by thestandard peroxidase enzymatic method. FIG. 27A showed a strong positivesignal of GD2 expression in all eight Rb samples and one of brain tumor,while there was no signal in normal brain tissue sample. The expressionof GD2 on Y79 retinoblastoma cell line was also investigated by flowcytometry. The results demonstrated that almost 100% of Y79 RB cellsexpressed GD2 (FIG. 27B). Additionally, low expression of GD2 was foundin primary neuroblastoma cells as previously reported. Expression ofCD19 protein was not found on Y79 cells and thus the CD19 protein wasused as a negative control along with CD19-specific CAR T cells forcomparison with the GD2-specific CAR T cells.

Generation of Primary T Cells Expressing GD2-Specific CAR

A lentiviral vector was generated encoding a GD2-specific CAR (GD2-CAR),which consisted of (a) anti-GD2 scFv, (b) the hinge and transmembraneregions of the CD8 molecule, (c) the CD28, 4-1BB and CD27 costimulatorysignaling moieties, and (d) the cytoplasmic component of CD3ζ molecule(FIG. 28A). Activated lymphocytes from a healthy donor were transducedwith lentiviral particles encoding the GD2-CAR or the CD19-CAR, whichwere used to generate negative control T cells. To determine whetherGD2-specific CARs were successfully transferred, the transduced cellswere detected for CAR expression on cell surface of T-cells by usingantibody. It was found that the CARs were expressed on the T-cellstransduced with the lentiviral particles.

Tumor Cell Lysis by GD2-Specific CAR T Cells

To test whether GD2-specific CAR T cells could kill the tumor cells, Y79RB wasort (T) were co-cultured with CD19- or GD2-specific CART cells(E). Cell death was analyzed by using AnnexinV/PI staining asdemonstrated in schematic diagram FIG. 28B. Co-culture experiment wasperformed at various E:T ratio to determine killing efficiency of CAR Tcells. It was found that GD2-specific CAR T cells induced cell death(FIG. 29A, 29B) and specific lysis of the target cells (FIG. 29C).Killing of target Rb cells was observed both on day 1 of co-culture andday 3 of co-culture with the GD2-CAR T cells (FIG. 29D). Some killingwas also observed at day 6 and day 14 of co-culture, although the degreeof killing was less than on day 1 or day 3 (FIG. 30). Overall, theGD2-CAR T cells were able to effectively kill the Rb cells.

It was determined that GD2 was downregulated in tumor cells upon GD2-CART cell co-culture (FIG. 31A) and that GD2-resistant Rb cells developed(FIG. 31B). It was also shown that PD-L1 expression increased uponGD2-CAR T cell co-culture (FIG. 31C). This may indicate that treatmentwith PD-L1 inhibitors or PD1 inhibitors may increase efficacy of theGD2-CAR T cells. Overall, however, the GD2-CAR T cells were able toeffectively kill the Rb cells, indicating that such T cells are usefulfor treating Rb, either alone or potentially in combination with a PD-L1or PD1 inhibitor.

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present disclosure, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the disclosure to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

EQUIVALENTS

While several inventive embodiments have been described and illustratedherein, those of ordinary skill in the art will readily envision avariety of other means and/or structures for performing the functionand/or obtaining the results and/or one or more of the advantagesdescribed herein, and each of such variations and/or modifications isdeemed to be within the scope of the inventive embodiments describedherein. More generally, those skilled in the art will readily appreciatethat all parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the inventive teachingsis/are used. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific inventive embodiments described herein. It is,therefore, to be understood that the foregoing embodiments are presentedby way of example only and that, within the scope of the appended claimsand equivalents thereto, inventive embodiments may be practicedotherwise than as specifically described and claimed. Inventiveembodiments of the present disclosure are directed to each individualfeature, system, article, material, kit, and/or method described herein.In addition, any combination of two or more such features, systems,articles, materials, kits, and/or methods, if such features, systems,articles, materials, kits, and/or methods are not mutually inconsistent,is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

All references, patents and patent applications disclosed herein areincorporated by reference with respect to the subject matter for whicheach is cited, which in some cases may encompass the entirety of thedocument.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03. It should be appreciatedthat embodiments described in this document using an open-endedtransitional phrase (e.g., “comprising”) are also contemplated, inalternative embodiments, as “consisting of” and “consisting essentiallyof” the feature described by the open-ended transitional phrase. Forexample, if the disclosure describes “a composition comprising A and B”,the disclosure also contemplates the alternative embodiments “acomposition consisting of A and B” and “a composition consistingessentially of A and B”.

1-63. (canceled)
 64. A monospecific chimeric antigen receptor (CAR)comprising: an antigen binding domain that is monospecific for CD19; atransmembrane domain, wherein the transmembrane domain comprises a CD28transmembrane domain; and a cytoplasmic domain comprising a 4-1BBintracellular domain, a CD27 intracellular domain, a CD3zeta signaltransduction domain, and an inducible caspase 9 domain.
 65. Themonospecific CAR of claim 64, wherein the antigen binding domain is asingle-chain variable fragment (scFv).
 66. The monospecific CAR of claim64, where the antigen binding domain comprises the amino acid sequencesset forth in SEQ ID NOs: 11 and
 12. 67. The monospecific CAR of claim64, wherein the CD28 transmembrane domain comprises the amino acidsequence set forth in SEQ ID NO: 23, 24, or
 25. 68. The monospecific CARof claim 64, where the 4-1BB intracellular domain comprises the aminoacid sequence set forth in SEQ ID NO:
 42. 69. The monospecific CAR ofclaim 64, wherein the CD27 intracellular domain comprises the amino acidsequence set forth in SEQ ID NO:
 38. 70. The monospecific CAR of claim64, wherein the CD3zeta signal transduction domain comprises the aminoacid sequence set forth in SEQ ID NO: 39 or
 40. 71. The monospecific CARof claim 64, wherein the inducible caspase 9 (iCasp9) domain comprisesthe amino acid sequence set forth in SEQ ID NO: 28 or
 29. 72. Themonospecific CAR of claim 71, further comprising an FKBP binding proteincomprising the amino acid sequence set forth in SEQ ID NO:
 30. 73. Animmune cell comprising the monospecific CAR of claim 64, wherein theimmune cell is a T cell or NK cell.
 74. A composition comprising aplurality of the immune cell of claim
 73. 75. A method comprisingadministering the immune cell of claim 73 to a subject having cancer.76. The method of claim 75, wherein the subject is a human.
 77. Themethod of claim 75, wherein the cancer is leukemia.
 78. The method ofclaim 77, wherein the leukemia is B cell leukemia.